Lymphoid chemokines in the diagnosis, monitoring and treatment of inflammatory disease

ABSTRACT

Experimental autoimmune encephalomyelitis (EAE) is a Th1-mediated autoimmune disease of the central nervous system that is widely used as an animal model of multiple sclerosis (MS). In this study it was demonstrate that CXCL13, a chemokine involved in the development of secondary lymphoid tissues, is expressed in CD11c+ myeloid cells that accumulate in EAE lesions. Blockade or deficiency of CXCL13 ameliorates clinical EAE, both during acute and relapsing stages. CXCL13 deficiency did not inhibit the priming or differentiation of autoimmune effector T-cells in the periphery, but appeared to exert its effects during the effector phase of pathogenesis. These findings indicate that reagents that antagonize or inhibit CXCL13 are useful for the treatment of neuroinflammatory diseases such as MS.

This application claims benefit of U.S. Provisional Application No.60/566,337, filed on Apr. 29, 2004, which is incorporated herein byreference in its entirety.

This invention was made with government support under NationalInstitutes of Health Grant NS41562. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

1. The majority of autoimmune diseases are chronic conditions,characterized by persistent or relapsing inflammation in the targetorgan. This is certainly true of multiple sclerosis (MS), aninflammatory disease of central nervous system (CNS) white matter, whichgenerally presents with recurrent episodes of neurological dysfunctionfollowed by a secondary stage of gradually worsening disability.Experimental autoimmune encephalomyelitis (EAE), an animal model withstrong pathological similarities to MS, also follows a relapsing,progressive clinical course (Raine, C. S., et al. 1984. LaboratoryInvestigation 51:534-546).

2. In the human disease multiple sclerosis (MS) and the animal model,experimental autoimmune encephalomyelitis (EAE), chronic or relapsinginflammation in the central nervous system (CNS) results in destructionof the myelin sheath. EAE is induced in naïve mice by the adoptivetransfer of CD4⁺ T-cells specific for myelin proteins (Raine, C. S., etal. 1984. Laboratory Investigation 51:534-546). Once these effectorcells cross the blood-brain-barrier, they activate resident microgliaand recruit peripheral leukocytes to the CNS (Bauer, J., et al. 1995.Glia 15:437). Perivascular infiltrates are subsequently formed withinthe white matter and evolve into lymphoid-myeloid aggregates over time.

3. In acute lesions, myeloid cells dominate the inflammatory infiltrate(Bauer, J., et al. 1995. Glia 15:437). T-cells become more numerous inchronic lesions and are sometimes found in association withdendritic-like (dendriform) cells. B cells and plasma cells are alsofrequently present, and tend to localize in regions distinct from thosecontaining T-cells (Raine, C. S., et al. 1984. Laboratory Investigation51:534-546, Paterson, P. Y., and Swanborg, R. H. 1988. In: ImmunologicalDiseases (ed. Samter, M.), Traugott, U., et al. 1983. Journal ofNeuroimmunology 4:201-221). Even lymphatic capillaries have beendemonstrated in the CNS tissues of patients with long standing MS(Prineas, J. W., and Wright, R. G. 1978. Laboratory Investigation38:409-421). The structured appearance and cellular composition of thesechronic CNS lesions are reminiscent of secondary lymphoid tissues.Similarly, in EAE white matter infiltrates exhibit features normallyassociated with lymph nodes and spleen including perivascular T-cellcuffs (approximating periarterial sheaths in the spleen),lymphoid-myeloid aggregates, and blood vessels with characteristics ofhigh endothelial venules (HEV).

4. Several features of chronic inflammatory infiltrates in autoimmunelesions parallel the architectural characteristics of secondary lymphoidtissues. For example, T and B cells tend to segregate into distinctareas of chronic MS plaques, similar to the T-cell-and B cell-rich areasof lymph node and spleen (Raine, C. S., et al. 1984. LaboratoryInvestigation 51:534-546, Paterson, P. Y., and Swanborg, R. H. 1988. In:Immunological Diseases (ed. Samter, M.)-3). Lymphatic-like capillarieshave been identified in long-standing MS lesions (Prineas, J. W., andWright, R. G. 1978. Laboratory Investigation 38:409-421). Furthermore,myeloid dendritic cells, a critical class of antigen presenting cells(APC) that serve to activate T-cells in peripheral lymphoid organs, haverecently been detected in MS and EAE lesions (Ulvestad, E., et al. 1994.J Leukoc Biol. 56:732-40, Fischer, H. G. and Reichmann, G. 2001. JImmunol. 166:2717, (Serafini, B., et al. 2000. American Journal ofPathology 157:1991-2002). Interestingly, microglia acquiredendritic-like cell characteristics following stimulation with GM-CSF invitro, raising the possibility that dendritic cells differentiate fromCNS glial cells in vivo (Fischer, H. G. and Reichmann, G. 2001. JImmunol. 166:2717, Santambrogio, L., et al. 2001. PNAS 98:6295, Aloisi,F., et al. 2000. J. Immunol. 164:1705). Although these studies suggestthat autoimmune infiltrates evolve using similar pathways to those thatguide the development and organization of secondary lymphoid tissues,needed in the art are specific agents that modulate relevant pathwaysinvolved in autoimmune disease.

5. CXCL13, also known as B-lymphocyte chemoattractant (BLC),B-cell-attracting chemokine 1 (BCA1), and Angie-2, is a protein of about88 amino acids in the group of CXC-Chemokines. The receptor for CXCL13is CXCR5 (also known as BLR-1 and MDR15), although CXCR3 has also beenidentified as a receptor.

SUMMARY OF THE INVENTION

6. In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates to anmethods of screening for agents that inhibit T-cell mediatedinflammatory responses. Also provided herein are methods of treating aninflammatory condition comprising administering an agent identified bythe disclosed screening methods.

7. Additional advantages of the invention will be set forth in part inthe description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

8. The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain theprinciples of the invention.

9. FIG. 1 shows that CXCL13 and CCL19 are expressed in the CNS of micewith EAE. FIG. 1(a) shows the results of an experiment in which B10.PLmice (n=5) were immunized with MBP_(Ac1-11) in CFA and sacrificed fiveweeks later during clinical EAE (one mouse remained asymptomatic).Spinal cords were harvested for RNA extraction and RT-PCR analysis.Cords from naïve mice (n=4) were used as negative controls. FIG. 1 (b)shows similar results using an adoptive transfer paradigm. In theexperiment shown, draining LN cells from PLP₁₃₉₋₁₅₁/IFA-sensitized SJLmice were injected into naïve syngeneic recipients (50×10⁶ i.p.)following in vitro challenge with antigen in the presence (left panel)or absence (right panel) of recombinant murine IL-12. Mice weresacrificed between days 10 and 12 post-transfer for RT-PCR analysis ofwhole spinal cord specimens. FIG. 1(c) shows that CXCL13 and CCL19proteins are expressed in spinal cord tissue from SJL mice withadoptively transferred EAE but not in spinal cords from their naïvecounterparts (n=10/group). Spinal cord tissues pooled from each groupwere analyzed for CXCL13, CCL19 and CCL21 protein expression by Westernblot. Spleen extracts and recombinant murine chemokines were used aspositive controls. All experiments were repeated at least two times withsimilar results.

10. FIG. 2 shows that lymphoid chemokine expression rises steadily inthe CNS during the course of relapsing-remitting EAE. By contrast,inflammatory chemokine levels fluctuate in concert with clinical score.SJL mice were immunized with PLP₁₃₉₋₁₅₁/CFA. Representative animals weresacrificed during successive stages of relapsing EAE for analysis oflymphoid (a) and inflammatory (b) chemokines by RPA. Chemokine levelswere quantified by phosphorimaging (lower panels). The results shown arerepresentative of 2 independent experiments.

11. FIG. 3 shows that CXCR5+CD4+ T-cells with a memory effectorphenotype, and a subset of CD4+CD3− cells, accumulate in the spinalcords of mice with EAE. FIG. 3(a) shows FACS analysis of spinal cordlymphoid cells (gated based on forward and side scatter characteristics)isolated from PLP₁₃₉₋₁₅₁/CFA-sensitized SJL mice following the firstepisode of clinical EAE. FIG. 3(b) shows CNS-infiltrating CD4+ CXCR5+and CXCR5− cells that were triple stained against a panel of activationmarkers to generate the histograms. Positive gates were determined basedupon background staining with isotype matched control antibodies. Theseexperiments were performed multiple times with similar results.

12. FIG. 4 shows that lymphoid chemokine transcripts are expressed inspinal cord mononuclear cells (MNCs) from mice with acute and relapsingEAE but not from naïve controls. Furthermore, CXCL13 mRNA is enriched inthe CD11c+ subpopulation. In the experiment shown in FIG. 4(a) SJL micewere immunized with PLP/CFA. Spinal cords were harvested during the 1stand 2nd episodes of EAE, respectively, to isolate infiltrating MNC byPercoll gradient centrifugation. MNCs were used for flow cytometricanalysis to characterize myeloid subsets and for extraction of RNA tomeasure expression of CXCL13, CCL19, CCL21 and lysozyme M (a myeloidcell marker) by RT-PCR. In FIG. 4(b), spinal cord MNCs from C57BL/6 micewith MOG₃₅₋₅₅-induced EAE or naïve controls were analyzed by FACS andRT-PCR as described above. In FIG. 4(c), spinal cord MNCs from C57BL/6mice with EAE were separated based on expression of the dendritic cellmarker, CD11c, using magnetic beads. Semiquantitative RT-PCR andsouthern blot hybridization were performed on unfractioned,CD11c-depleted and CD11c-enriched fractions as shown. Relative mRNAlevels were determined by phosphorimaging and densitometry.

13. FIG. 5 shows CXCL13 deficiency or blockade supresses EAE during theeffector stage. In the experiments shown in FIG. 5(A), EAE was inducedin C57BL/6 wildtype (open circles; n=20) and CXCL13−/− (closed circles;n=19) mice by active immunization with MOG₃₅₋₅₅/CFA. The difference inclinical scores between groups is statistically significant (P<0.0001 bythe Mann-Whitney Rank Sum Test). The results are representative of threeexperiments. In the experiments shown in FIG. 5(B), EAE was induced inC57BL/6 wildtype (open circles; n=8) and CXCL13−/− (closed circles; n=9)mice by adoptive transfer and rated as described above. The differencein clinical scores between groups is statistically significant (P<0.0001by the Mann-Whitney Rank Sum Test). This experiment was performed twicewith similar results. FIG. 5(C) shows the clinical courses of SJL micethat were injected i.p. with PLP₁₃₉₋₁₅₁-reactive LN cells on day 0 andeither goat anti-mouse CXCL13 antibody (0.2 mg, R&D), control goat IgG(0.2 mg) or PBS on days 3, 6, and 10 (n=5/group). Statisticaldifferences were observed between the anti-CXCL13 treated group and eachof the control groups (P<0.05, Student-Newman-Keuls method) but notbetween the two control groups. FIG. 5D shows that EAE was induced inC57BL/6 WT and CXCL13−/− mice by adoptive transfer of purified CD4+T-cells from primed WT donors. Clinical scores reflect viable animalsonly; mice that died of EAE (3/5 in the WT group; 2/7 in the CXCL13−/−group) were given a score of 5 on the day of death. The difference inclinical scores between groups is statistically significant (P<0.0001).The experiment was performed 3 times with similar results.

14. FIG. 6 shows that CXCL13−/− mice with EAE exhibit relatively mildhistopathological changes in the CNS. Spinal cord sections from C57BL/6CXCL13−/− and wildtype mice sacrificed during the first exacerbation (a)or later stages (b) of EAE were fixed in paraformaldehyde, embedded inparaffin and stained with the indicated reagents. Representativesections are shown. Original magnification: ×4 for H&E; ×10 for allother stains.

15. FIG. 7 shows that CD11b+myeloid cells are disproportionatelydepleted in CNS infiltrates of CXCL13−/− mice. C57BL/6 wildtype andCXCL13−/− mice were immunized with MOG₃₅₋₅₅. Spinal cord MNCs wereisolated during EAE relapse or remission for FACS analysis. Cells werestained with αCD11b (filled histogram) or isotype control (solid line)antibodies. The percentage of CD11b+ cells and median flouresenceintensity (MFI) are shown.

16. FIG. 8 shows that CD11c⁺ cells are present in EAE infiltrates andform clusters with infiltrating T-cells. FIG. 8(A) shows a frozensection of a spinal cord from a representative SJL mouse with EAE(clinical score 3). The section was stained with FITC conjugatedanti-CD4 and PE-conjugated anti-CD11c and analyzed on an MRC-600confocal laser microscope system The lower panel is of the same sectionphotographed using a filter that screens out the FITC-signal in order tohighlight the staining for dendritic-like cells. FIG. 8(B) shows aperivascular infiltrate in the spinal cord of a C57BL/6 mouse with EAE(score 3) that was visualized by whole mount technique. In this imageCD4+ cells were stained with an APC conjugated antibody, dendritic-likecells with PE-conjugated anti-CD11c and inflamed blood vessels withFITC-conjugated anti-P-selectin. Spinal cords from naïve mice failed tostain with any of the antibodies. Background from isotype controlantibodies was negligible.

17. FIG. 9 shows that Encephalitogenic PLP-primed LN cells contain asubpopulation of CXCR5⁺ CD3⁺ T-cells. Draining LN cells from 10 PLP/CFAimmunized SJL mice were resected on day 10, pooled and cultured withantigen. The cells were harvested at 96 h, washed, permeabilized, andstained with labeled anti-CXCR5 monoclonal antibodies or with isotypematched control antibodies, as indicated. Naïve LN cells do not containdetectable CD3⁺ CXCR5⁺ cells.

18. FIG. 10 shows that MAdCAM is upregulated on cerebrovasculatureduring EAE. Pictured is an EAE lesion in the spinal cord of aMOG-sensitized C57BL/6 mouse with EAE (clinical score 2). A whole mountsection of the thoracolumbar cord was stained with anti-CD45-FITC andanti-MAdCAM-PE prior to visualization under a fluorescent microscope.Isotype controls showed negligible background staining. Anti-MADCAM doesnot stain vessels in spinal cords from naïve or mock-immunized mice.

19. FIG. 11 shows the circle transcripts and AID mRNA are present inspinal cords of mice with EAE. Spinal cords were removed fromPBS-perfused mice with EAE or naïve controls. RNA was extracted andRT-PCR performed with primers for AID (A) or CTγ3 (B), followed bySouthern Blot Hybridization with internal oligonucleotide probes (forAID or CTγ3, respectively).

20. FIG. 12 shows that CXCL13−/− mice generate myelin reactiveencephalitogenic T-cells that initiate CNS inflammation. FIG. 12A showsC57BL/6 WT and CXCL13−/− mice that were immunized with MOG₃₅₋₅₅/CFA.Spleens were harvested to assess antigen-specific proliferation (day 19post immunization) and cytokine production (data from individual mice;days 24-27). The ELISPOT data show antigen-specific responses (50 μg/mlof MOG). The results are representative of two or more experiments. (*P<0.05). FIG. 12 B shows splenocytes from MOG/CFA primed CXCL13−/− or WTmice were stimulated in vitro with MOG and IL-12, and injected into WTrecipients. The difference in clinical scores between groups isstatistically significant (P<0.0001). The experiment shown was performedthree times with similar results.

21. FIG. 13 shows that CNS-infiltrating T-cells are diminished inCXCL13-deficient mice during late stages of EAE. C57BL/6 CXCL13−/− andWT mice were immunized with MOG₃₅₋₅₅/CFA. FACS analysis of pooled CNSMNCs was performed during the 1st episode of EAE (days 15-21), or duringremission (day 42). FIG. 13A shows that the absolute number ofinfiltrating CD4+ T-cells/cord was calculated by multiplying theirpercentage within the inflammatory infiltrate (determined using FACS) bythe total number of MNCs per cord. Results shown represent the mean oftwo representative experiments. FIG. 13B shows dot plots that weregenerated using the lymphoid scatter gate. All experiments wereperformed three or more times with similar results.

22. FIG. 14 shows that myeloid cells are disproportionately depleted inCNS infiltrates of CXCL13−/− mice during late stages of EAE. C57BL/6 WTand CXCL13−/− mice were immunized with MOG₃₅₋₅₅/CFA. Spinal cord MNCswere isolated during the 1st episode or remission of EAE for FACSanalysis. FIG. 14A shows dot plots that were generated using a widescatter gate to include all viable cells. FIG. 14B shows the absolutenumbers for CD45^(hi) CD11b^(hi) cells in the CNS infiltrate calculatedas described in the legend to FIG. 13. The numbers are derived from therepresentative experiments shown in (A). These experiments were repeatedthree times with similar results.

23. FIG. 15 shows that administration a monoclonal antibody againstCXCL13 ameliorates adoptively transferred EAE. EAE was induced in naïveB10.PL female mice (n=5/group) by transfer of splenocytes from B10.PLdonors that express a T-cell receptor transgene specific for myelinbasic protein (MBP-TCR). MBP-TCR splenocytes were stimulated withantigen (MBP peptide Ac1-17; 50 μg/ml)) and recombinant murine IL-12 (2ng/ml) for 96 hours prior to transfer (35×10⁶ cells/recipient).Recipients were injected with monoclonal antibodies (either anti-CXCL13or control rat IgG2a, R&D; 300 μg i.p.) on days 3, 6 and 10 post celltransfer. Mice in both groups were rated for degree of paralysis on a 5point scale by an examiner who was blinded to treatment group. * p=0.03by Whitney-Mann test.

DETAILED DESCRIPTION

24. The present invention may be understood more readily by reference tothe following detailed description of preferred embodiments of theinvention and the Examples included therein and to the Figures and theirprevious and following description.

25. Before the present compounds, compositions, articles, devices,and/or methods are disclosed and described, it is to be understood thatthis invention is not limited to specific synthetic methods, specificrecombinant biotechnology methods unless otherwise specified, or toparticular reagents unless otherwise specified, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to be limiting.

Definitions

26. As used in the specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apharmaceutical carrier” includes mixtures of two or more such carriers,and the like.

27. Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

28. In this specification and in the claims which follow, reference willbe made to a number of terms which shall be defined to have thefollowing meanings:

29. “Optional” or “optionally” means that the subsequently describedevent or circumstance may or may not occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not.

30. The term “subject” is used throughout this disclosure to refer toany organism, tissue, or cell being contacted with the agent or treatedwith the agent. Such subjects include but are not limited to tissueculture cells, mammals, mice, rats, guinea pigs, dogs, pigs, rabbits,sheep, monkeys, chimpanzees, and humans. It is understood and hereincontemplated that the disclosed methods of screening include methods ofscreening, wherein the subject is a mammal. It is also understood thatthe disclosed methods of treatment include methods of treatment whereinthe subject is a mammal. In one embodiment, the subject is a mammal, forwhom diagnosis or therapy is desired. In a specific embodiment, thesubject is a human for whom diagnosis or therapy is desired.

31. Herein “inhibition,” “inhibits,” or “inhibiting” refer to themodulation of a cell, interaction, or action in a reducing manner. It isunderstood that “inhibition” can refer to any decrease in an action oractivity of a cell, or as cellular interaction, or molecular interactioninteraction, or action including but not limited to the completeablation and/or prevention of the action, interaction, or activity. Forexample, inhibition of T-cell mediated inflammatory responses includesdecreasing the degree of the inflammatory response by 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% or any point in between, comparedto an untreated or control subject as determined by any suitablemeasurement technique or assay disclosed herein or known in the art.Furthermore, the untreated or control subject can be a separate subjectmeasured at the same or a different time as the treated subject, or thetreated subject measured prior to treatment, with the pre-treatmentvalues compared to the post-treatment values to determine the level ofreduction. Thus, for example an agent that inhibits a T-cell mediatedinflammatory response refers to any agent that can decrease T-cellmediated inflammation by as little as 5% of the total inflammation aswell as agents that completely ablate the inflammatory response. Any ofthese treatment types or types of patients may also be excluded.

32. “Agent” refers to any composition including but not limited toantibodies, siRNA, chemical compositions, cytokines, chemokines, orsmall molecules. The agents of the invention can be prepared aspharmaceutical compositions and combined with adjuvants to increasetheir effect. For example, the agent can comprise an antibody thatblocks the action of CXCL13. Thus also disclosed are methods, whereinthe agent to be screened is a neutralizing antibody to CXCL13.Similarly, the agents may also comprise antibodies to other chemokinesor chemokine receptors. Therefore, one embodiment of the disclosedmethods are methods, wherein the agent is an antibody to CXCR5 andwherein the antibody blocks CXCL13 binding without causing signalingthrough CXCR5. It is understood that the disclosed agents can compriseboth membrane bound and soluble forms of chemokines, cytokines, ligands,and their receptors. Thus, for example, specifically contemplated aremethods, wherein the agent is a soluble form of CXCR5 or a derivative oranalog thereof. In another embodiment, the agent may be an analog orderivative of CXCL13 (e.g., a dominant negative form of CXCL13 or someother form of CXCL13 which is modified from the normal, native formCXCL13) which blocks binding of endogenous CXCL13 to its receptors(e.g., CXCR5 and/or CXCR3), or otherwise interferes with receptorfunctions which functions include, without limitation, the ability tobind ligand molecules, the ability to interact with other proteins, theability to generate a “signal” affecting the properties or behaviors ofthe cell expressing the receptor, or the ability to interact with oraffect other cells. Any of these agents may also be excluded. Thus, forexample, in one embodiment, the agent of the present invention is not ansiRNA, small organic molecule, or macromolecule.

33. The terms “treat,” “treatment,” or “treating” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented. For example, treating aninflammatory condition means reducing the extent or severity of theinflammation. The reduction can mean but is not limited to the completeablation of inflammation. For example, the reduction can comprise a 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction, or anypoint in between, compared to an untreated or control subject asdetermined by any suitable measurement technique or assay disclosedherein or known in the art. Furthermore, the untreated or controlsubject can be a separate subject measured at the same or a differenttime as the treated subject, or the treated subject measured prior totreatment, with the pre-treatment values compared to the post-treatmentvalues to determine the level of reduction. Any of these treatment typesor types of patients may also be excluded.

34. By “specifically binds,” it is generally meant that an antibodybinds to an epitope via its CDR, and that the binding entails somecomplementarity between the CDR and the epitope. According to thisdefinition, an antibody is said to “specifically bind” to an epitopewhen it binds to that epitope, via its CDR more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

Methods of Screening

35. Experimental autoimmune encephalomyelitis (EAE) is an inflammatorydemyelinating disease of CNS white matter that frequently follows arelapsing or progressive clinical course. It is widely used as an animalmodel of multiple sclerosis (MS). Adoptive transfer studies havedemonstrated that myelin-reactive CD4+ Th1 cells trigger the diseaseprocess in EAE (Ando, D. G., et al. 1989. Cell Immunol 124:132-143).However, non-specific lymphocytes and myeloid cells constitute themajority of cells in established CNS infiltrates and are largelyresponsible for the end organ damage (demyelination and axonal injury)that results in neurological deficits (Bauer, J., et al. 1996.Histochemical Journal 28:83-97, Cross, A. H., et al. 1990. Lab Invest63:162-170, (Raine, C. S., et al. 1990. Lab Invest 63:476-489,(Skundric, D. S., et al. 1993. Journal of Neuroimmunology 46:113-121).These inflammatory cells are not randomly distributed in the CNS butform discrete perivascular infiltrates in subpial and parenchymal whitematter tracts. Throughout the course of disease, myelin-reactive T-cellsremain adjacent to blood vessels and are surrounded by macrophages andactivated microglia that extend into the parenchyma (Raine, C. S., etal. 1990. Lab Invest 63:476-489, (Raine, C. S., et al. 1990. ClinicalImmunology & Immunopathology 57:173-187). Many laboratories haveinvestigated the role of “pro-inflammatory” chemokines, such as CCL2,CCL3, CCL5 and CXCL10, in attracting circulating leukocytes to acutedemyelinating lesions (Fife, B. T., et al. 2001. J Neurosci Res66:705-714, Karpus, W. J., and Ransohoff, R. M. 1998. Journal ofImmunology 161:2667-2671).

36. Certain histological features of established MS and EAE lesions arereminiscent of secondary lymphoid tissues. Such features includeperivascular T-cell cuffing, clustering of T-cells and dendritic-likecells into lymphoid-myeloid aggregates, and the segregation of B andT-cells into distinct areas within chronic plaques (Cross, A. H., et al.1990. Lab Invest 63:162-170, Traugott, U., et al. 1981. Science214:1251-1253, Traugott, U., et al. 1983. Journal of Neuroimmunology4:201-221, Raine, C. S., et al. 1980. Laboratory Investigation43:150-157, Raine, C. S., et al. 1984. Laboratory Investigation51:534-546, Prineas, J. W., and Wright, R. G. 1978. LaboratoryInvestigation 38:409-421, Prineas, J. W. 1979. Science 203:1123-1125,Pashenkov, M., et al. 2003. Brain Pathology 13:23-33, Serafini, B., etal. 2000. American Journal of Pathology 157:1991-2002). In aneuropathological survey of demyelinating lesions in SJL mice withrelapsing EAE, Raine and colleagues observed “nests of smalllymphocytes, large mononuclear cells and plasma cells separated by septafrom reticular-like cells giving the tissue a sinusoidal arrangement”(Raine, C. S., et al. 1984. Laboratory Investigation 51:534-546).Similarly, Prineas described “clusters of plasma cells together with . .. reticular cells surrounding collagen-free channels containinglymphocyes and macrophages” in plaques in brain specimens from patientswith MS (Prineas, J. W. 1979. Science 203:1123-1125). Lymphatic-likecapillaries were also identified in long standing MS lesions. In both ofthe cited references parallels were drawn between the organization ofthe CNS infiltrates and the medullary regions of a lymph node. Analogieshave also been made between the vascular elements in inflammatorydemyelinating lesions and lymphoid tissues. For example, endothelialcells in inflamed blood vessels of chronic EAE plaques were noted to beenlarged, cuboidal and to bulge into the vessel lumen, evocative of highendothelial venules (HEVs) in LNs (Raine, C. S., et al. 1990. Lab Invest63:476-489). MAdCAM-1 and MECA-325, adhesion molecules normallyrestricted to HEV, were detected on CNS blood vessels in mice afflictedwith EAE (Raine, C. S., et al. 1990. Clinical Immunology &Immunopathology 57:173-187, Vissers, J. L. M., et al. 2001. Eur. J.Immunol. 31:1544, Luther, S. A., et al. 2000. Immunity 12:471-481).Thus, “lymphoid neogenesis,” or the formation of new lymphoid tissues,occurs within the brain and spinal cord during the evolution of EAE andMS.

37. Chemokines play a pivotal role in leukocyte migration. During acuteEAE, “inflammatory” chemokines such as RANTES, MIP1α, MIP1β and MCP-1are expressed in the spinal cord and presumably contribute to theaccumulation and activation of mononuclear cells bearing CCR5 and CCR2receptors (Karpus, W. J. and Ransohoff, R. M. 1998. J. Immunol. 161:2667). Similarly, RANTES and MIP1α have been detected in active whitematter lesions in autopsy specimens from patients with MS (Boven, L. A.,et al. 2000. Clin Exp Immunol. 122:257, (Simpson, J. E., et al. 1998. JNeuroimmunol. 84:238).

38. Lymphoid chemokines, including CXCL13 (BLC), CCL19 (ELC), and CCL21(SLC), are critical for the formation of peripheral lymphoid organs.They guide the migration of leukocyte subsets to B cell and T-cell richareas (Cyster, J. G. 1999. Science 286:2098-2102, Moser, B., andLoetscher, P. 2001. Nature Immunol. 2:123). CXCL13 is produced bystromal cells in lymphoid follicles and interacts with the CXCR5receptor, which is expressed on B cells and a subpopulation of T-cells(Gunn, M. D., et al. 1998. Nature. 391:799, Legler, D. F., et al. 1998.J. Exp. Med. 187:665, Kim, C. H., et al. 2001. J Exp Med.193:1373-1381). CXCR3 has also been identified as a receptor for CXCL13(Jenh, C. H. et al., Cytokine 15:113-121 (2001). CCL19 and CCL21 areproduced by high endothelial venules and stromal cells in T-cell richareas. They attract T-cells and mature dendritic cells bearing thereceptor CCR7 (Campbell, J. J., et al. 1998. J. Cell. Biolo. 141:1053,Luther, S. A., et al. 2002. J Immunol 169:424-433). The expression ofeach of the lymphoid chemokines is dependent, in large part, onLymphotoxin (LT)-α/β and TNFα (Hjelmstrom, P., et al. 2000. Am J Path156:1133-1138, Ngo, V. N., et al. 1999. J. Exp. Med. 189:403). Of note,(LT)-α/β and TNFα have been implicated in the pathogeneisis of organspecific autoimmune diseases, including EAE and MS (Selmaj K, et al.1995. Neuroimmunol. 56(2):135, Suen, W. E., et al. 1997. J. Exp. Med.186:1233, Eugster, H-P, et al. 1998. Eur. J. Immunol. 29:626).

39. CXCL13 and CCL19 can also be produced by subpopulations of myeloiddendritic cells (Vissers, J. L. M., et al. 2001. Eur. J. Immunol.31:1544, Forster, R., et al. 1996. Cell 87:1037-1047). This isparticularly relevant since myeloid cells expressing a dendriticcell-like (dendriform) morphology and cell surface phenotype have beendetected in inflamed CNS white matter, including specimens harvestedfrom mice with EAE (FIG. 9) (Fischer, H. G. and Reichmann, G. 2001. JImmunol. 166:2717, (Serafini, B., et al. 2000. American Journal ofPathology 157:1991-2002). There is growing evidence that such dendriformcells differentiate from microglial precursors. For example, microgliadifferentiate into dendriform cells in vitro following stimulation withGM-CSF (Fischer, H. G. and Reichmann, G. 2001. J Immunol. 166:2717,Santambrogio, L., et al. 2001. PNAS 98:6295, Aloisi, F., et al. 2000. J.Immunol. 164:1705). GM-CSF is produced in spinal cords during EAE, mostlikely by infiltrating effector T-cells (Wong, R. L., et al. 1989. Cell.Immunol. 123:445).

40. Transgenic expression of CXCL13 or CCL21 in pancreatic isletstrigger the local formation of organized lymphoid structures (Luther, S.A., et al. 2000. Immunity 12:471-481, Fan, L., et al. 2000. J. Immunol.164:3955, Luther, S. A., et al. 2002. J Immunol 169:424-433).Furthermore, endogenous lymphoid chemokines have been detected in thetarget organs of patients with autoimmune diseases as well as at sitesof chronic infection. For example, CXCL13 was found in the salivaryglands of patients with Sjogren's syndrome, the synovial tissue ofpatients with rheumatoid arthritis, and in gastric mucosal tissue in thesetting of refractory H. pylori infection (Salomonsson, S., et al. 2002.Scan J Immunol 55:336-342, Shi, K., et al. 2001. J Immunol 166:650-655,Mazzucchelli, L., et al. 1999. J. Clin. Inv. 104:R49-54, Takemura, S.,et al. 2001. J. Immunol. 167:1072). In addition, CXCL13 is expressed inthe thymus and kidneys of mice developing lupus nephritis (Ishikawa, S.,et al. 2001. J Exp Med 193:1393-1402), while CCL21 is expressed in thepancreas of NOD mice developing diabetes (Hjelmstrom, P., et al. 2000.Am J Path 156:1133-1138).

41. CXCR5 is most commonly expressed on B lymphocytes and CXCL13/CXCR5interactions are critical for stimulating germinal center reactions.Therefore it is not surprising that previous studies have concentratedon the role of CXCL13 in autoimmune diseases that are viewed asprimarily autoantibody driven, such as Sjogren's syndrome. By contrast,EAE is induced by autoreactive CD4⁺ T-cells. The inflammatory process inMS is also believed to be driven, to a great extent, by myelin-reactiveT-cells. Nonetheless, B cells and autoantibodies can participate inautoimmune demyelination, indicating that CXCL13 can be important insuch disorders. First, B cells accumulate in chronic MS and EAE plaquesover time. Furthermore, ninety percent of patients with MS developoligoclonal bands in their cerebrospinal fluid, indicative of localantibody production in the CNS. Analysis of the immunoglobulinrepertoire in spinal fluid and CNS biopsy specimens from MS patientssuggest that antigen-driven B cell clonal expansion and somatichypermutation occur within the target organ itself. Such phenomena arereminiscent of CXCL13-dependent germinal center reactions. Although therequirement for B cells in EAE varies based on the murine strain andautoantigen used for disease induction, autoantibodies against myelinantigens have been shown to facilitate demyelination in a wide varietyof rodent as well as non-human primate models. Circle transcripts andAID mRNA are present in spinal cords of mice with EAE indicated that Bcell differentiation occurs within the inflamed CNS (FIG. 11).Collectively, these data indicate that CXCL13 driven collaborationsbetween T and B cells can play an important role in diseases such as EAEand MS.

42. In addition to its effects on B cells, CXCL13 can play a role in EAEby modulating, and/or positioning a subset of CXCR5 expressing T-cellsin the CNS. The majority of naïve, OVA-specific CD4⁺ T-cells transientlyupregulate CXCR5 following immunization with antigen in CFA. CXCR5 isdetected on a significant percentage of CD4⁺ T-cells from myelin proteinimmunized donors immediately ex vivo (FIG. 9). CXCR5 is also expressedby a subset of iCOS⁺, IL-7R⁺ effector memory cells (T_(em)). Hence, aprimary role of CNS-produced CXCL13 in EAE can be the recruitment,positioning, and/or modulation of recently activated effector T-cellsand/or autoaggressive T_(em) in demyelinating plaques.

43. Herein it is disclosed that: (i) CXCL13, CCL19 and CCL21 aresecreted by CD11c⁺ myeloid cells in EAE lesions; (ii) CXCL13, CCL19 andCCL21 recruit, position, and/or modulate CXCR5⁺ and CCR7⁺ leukocytes,respectively, in EAE plaques and help shape the cellular composition ofwhite matter infiltrates; (iii) downstream effects of CNS lymphoidchemokines include the initiation of lymphoid neogenesis within plaques;(iv) CXCL13 also promotes B cell differentiation within the CNS, (v)CXCL13, CCL19 and CCL21 ultimately act to exacerbate the intensity andchronicity of clinical EAE, and; (vi) CXCL13 promotes relapses of EAE.The overarching principal is that ectopic production of lymphoidchemokines (such as CXCL13, CCL19 and CCL21) in the CNS drives theformation of organized inflammatory infiltrates characteristic ofchronic EAE and MS lesions and thereby promotes clinical relapses anddisease progression.

44. Lymphoid chemokines, including CCL19 (ELC), CCL21 (SLC) and CXCL13(BLC), are constitutively expressed in secondary lymphoid tissues andare responsible for the distinctive architecture of those organs (Ansel,K. M., et al. 2000. Nature 406:309-314, Luther, S. A., et al. 2003. JExp Med. 197:1191-1198). They direct leukocyte trafficking through thespecialized compartments of LNs and spleen (Moser, B., and Loetscher, P.2001. Nature Immunology 2:123-128, Cyster, J. G. 1999. Science286:2098-2102). CCL19 and CCL21 attract CCR7+ naïve and central memoryT-cells as well as activated dendritic cells to T-cell zones, whereasCXCL13 attracts CXCR5+ B cells and a subset of T helper cells (termedfollicular T helper cells) to B cell rich areas (Gunn, M. D., et al.1999. J. Exp. Med. 189:451-460, Kim, C. H., et al. 2001. J Exp Med.193:1373-1381, Forster, R., et al. 1996. Cell 87:1037-1047, Ansel, K.M., et al. 1999. J Exp Med. 190:1123-1134). Lymphoid chemokines areectopically expressed in nonlymphoid tissues during chronicinflammation. For example, CXCL13 was detected in gastric mucosal tissueand gastric lymphomas in the setting of refractory Helicobacter pyloriinfection (Mazzucchelli, L., et al. 1999. J Clin. Inv. 104:R49-54).CCL21 also plays a role in the development of lymphoid-like structureswithin the liver in response to Propionibacterium acnes infection thatwere critical for elimination of the bacteria (Yoneyama, H., et al.2001. J Exp Med 193:35-49). Furthermore, transgenic expression oflymphoid chemokines in the skin or pancreas drives the development oflymph node-like structures in those tissues (Luther, S. A., et al. 2000.Immunity 12:471-481)

45. While lymphoid chemokines might be expressed in non-lymphoid organsas part of an adaptive response against infection, these factors alsohave the potential to support chronic autoimmune inflammation. In fact,CCL19 and CCL21 are upregulated in the pancreas of NOD mice withdiabetes as well as in cerebrovasculature of mice with EAE (Hjelmstrom,P., et al. 2000. Am J Path 156:1133-1138, Alt, C., et al. 2002. Euro JImmunol 32:2133-2144, Columba-Cabezas, S., et al. 2003. Brain Pathology13:38-51). On the other hand, CXCL13 is expressed in the thymus andkidneys of mice developing experimental lupus nephritis (Ishikawa, S.,et al. 2001. J Exp Med 193:1393-1402). With regard to autoimmunediseases in humans, CXCL13 has been detected in salivary glands frompatients with Sjogren's syndrome and synovial tissues from patients withrheumatoid arthritis (Shi, K., et al. 2001. J Immunol 166:650-655,Salomonsson, S., et al. 2002. Scan J Immunol 55:336-342). CCL19 andCCL21 levels are elevated in cerebrospinal fluid from patients with MS,but not from patients with non-inflammatory neurological conditions(Pashenkov, M., et al. 2003. J Neuroimmunol 135:154-160). Nevertheless,prior to the present invention, the physiological role of endogenouslymphoid chemokines in the pathogenesis of these or other autoimmunediseases was not demonstrated.

46. Since CXCL13 is primarily known as an attractant for B cells andfollicular T helper cells, investigations on its role in autoimmunityhave largely been restricted to those diseases that are stronglyassociated with autoantibodies, such as Sjogren's syndrome and systemiclupus erythematosis (Ishikawa, S., et al. 2001. J Exp Med 193:1393-1402,35). EAE, on the other hand, is mediated by myelin-specific CD4+ Th1cells (Ando, D. G., et al. 1989. Cell Immunol 124:132-143). Th1 cellsare also believed to drive the disease process in MS (Voskuhl, R. R., etal. 1993. Autoimmunity 15:137-143, Bielekova, B., et al. 2000. Nat Med6:1167-1175). Nonetheless, several features of these autoimmunedemyelinating syndromes suggest that CXCL13 could contribute to theirpathogenesis. First, as mentioned above, infiltrates in demyelinatingplaques have been found to display features of secondary lymphoidtissues and CXCL13 is a key regulator of lymphoid neogenesis. Hence,transgenic expression of CXCL13 under the rat insulin promoter inducesthe formation of lymph node-like structures in the pancreas (Luther, S.A., et al. 2000. Immunity 12:471-481). Pancreatic infiltrates in thetransgenic mice are distinguished by a reticular stromal network,MAdCAM-1+ blood vessels and local induction of CCL21; all of thesecharacteristics have been observed in EAE and/or MS lesions (Cross, A.H., et al. 1990. Lab Invest 63:162-170, Raine, C. S., et al. 1980.Laboratory Investigation 43:150-157, Raine, C. S., et al. 1984.Laboratory Investigation 51:534-546, Prineas, J. W., and Wright, R. G.1978. Laboratory Investigation 38:409-421, Prineas, J. W. 1979. Science203:1123-1125, Kanwar, J. R., et al. 2000. J Neuroimmunol 103:146-152,Alt, C., et al. 2002. Euro J Immunol 32:2133-2144, Columba-Cabezas, S.,et al. 2003. Brain Pathology 13:38-51). Second, interactions between Tand B cells occur within the CNS during autoimmune demyelination andhave been implicated in MS and EAE pathogenesis (Baranzini, S. E., etal. 1999. J Immunol 163:5133-5144, Correale, J., and de los MilagrosBassani Molinas, M. 2002. J of Neurology 249:375-389, Colombo, M., etal. 2000. J Immunol 164:2782-2789, Cross, A. H., et al. 2001. JNeuroimmunol 112:1-14, Lyons, J. A., et al. 1999. European J Immunol29:3432-3439, Gerritse, K., et al. 1994. J Neuroimmunol 49:153-159,Genain, C. P., et al. 1995. Journal of Clinical Investigation96:2966-2974). Such interactions are likely to be facilitated by localCXCL13 production. Also, it is disclosed and herein contemplated thatCXCL13 can draw CXCR5+ effector or naïve T-cells that enter the CNStowards myeloid dendritic cells that, in turn, can act as antigenpresenting cells. This can result in efficient reactivation ofautoreactive T-cells within the target organ and/or priming of naïvemyelin-reactive T-cells and epitope spreading.

47. Thus, disclosed herein are methods of screening for an agent thatinhibits a T-cell mediated inflammatory response in a subject with aninflammatory response comprising administering to the subject the agentand detecting the presence of CXCL13 in the subject, wherein a reductionin the level of CXCL13 as compared to a control level indicates an agentthat inhibits the inflammatory response.

48. The disclosed methods comprise methods of screening for an agentthat inhibits a T-cell mediated inflammatory response. It is understoodand herein contemplated that “T-cell mediated inflammatory response”means a CD4 T-cell, NK T-cell, or CD8 T-cell response whose mode ofaction is the secretion of inflammatory cytokines. Such responses canoccur as the result of any type of immunological or physiological insultincluding but not limited to inflammatory conditions, viral infections,bacterial infections, yeast infections, parasitic infections, andcancers. T-cell mediated responses can be detected by numerousparameters including but not limited ELISA, ELISpot, and Flow cytometry(including, for example, Intracellular staining or CFSE staining),cytotoxicity assays (such as chromium release or JAM assays), andstandard lymphoproliferation assays (ie, involving incorporation oftritiated thymidine in vitro or BdRU in vivo).

49. For example the disclosed screening methods can comprise methods ofscreening for an agent that inhibits a T-cell mediated inflammatoryresponse, wherein the inflammatory response comprises a response to aninflammatory condition. Inflammatory conditions are well known in theart and can include autoimmune diseases. Thus also disclosed are methodsof screening for an agent that inhibits a T-cell mediated inflammatoryresponse, wherein the inflammatory response comprises a response to aninflammatory condition, wherein the inflammatory condition is selectedfrom the group consisting of asthma, alopecia greata, systemic lupuserythematosus (SLE), rheumatoid arthritis, reactive arthritis,spondylarthritis, systemic vasculitis, insulin dependent diabetesmellitus, multiple sclerosis, experimental allergic encephalomyelitis,Sjogren's syndrome, graft versus host disease, inflammatory boweldisease including Crohn's disease, ulcerative colitis, ischemiareperfusion injury, myocardial infarction, Alzheimer's disease,transplant rejection (allogeneic and xenogeneic), thermal trauma, anyimmune complex-induced inflammation, glomerulonephritis, myastheniagravis, cerebral lupus, Guillain-Barre syndrome, vasculitis, systemicsclerosis, anaphlaxis, catheter reactions, atheroma, infertility,thyroiditis, ARDS, post-bypass syndrome, hemodialysis, juvenilerheumatoid, Behcets syndrome, hemolytic anemia, pemphigus, bullouspemphigoid, stroke, atherosclerosis, scleroderma, psoriasis,sarcoidosis, transverse myelitis, acute disseminated encephalomyelitis,post-infectious encephalomyelitis, subacute sclerosing panencephalitis,and chronic inflammatory demyelinating polyradiculopathy. In oneembodiment, the inflammatory condition is multiple sclerosis. Each andany of these inflammatory conditions may also be excluded. Thus, forexample, in certain embodiments, the inflammatory condition is notasthma, not SLE, not rheumatoid arthritis, not myasthenia gravis, notdiabetes (e.g., not insulin dependent diabetes mellitus), or nottransplant rejection.

50. Also disclosed are methods of screening for an agent that inhibits aT-cell mediated inflammatory response, wherein the inflammatory responsecomprises a response to a viral antigen. Viral antigens are well knownin the art. It is understood that some of the pathogenic effects of aviral infection are not actually caused by the infecting virus, but bythe immune response to the virus. For example, LymphocyticChoriomeningitis virus (LCMV) will only induce pathogenic effects inhosts with functions T-cell responses. A host depleted of T-cells willshow no ill effects of an LCMV infection. However, an immunologicallyintact host will potentially suffer sever meningitis due to thecytolytic action and cytokine secretion of T-cells. Additionally, someviruses have been implicated in the pathogenesis of inflammatoryconditions such as multiple sclerosis. For example, Human Herpes virus-6(HHV-6), measles, and Epstein Barr virus (EBV) have been implicated inMS pathogenesis. Thus specifically disclosed are methods of screeningfor an agent that inhibits a T-cell mediated inflammatory response,wherein the inflammatory response comprises a response to HHV-6.Similarly, also disclosed are methods of screening for an agent thatinhibits a T-cell mediated inflammatory response, wherein theinflammatory response comprises a response to EBV or measles. Otherinflammatory conditions are also associated with viruses. For exampleHTLV-1 is associated with a myelopathy that presents similarly to MS.

51. Therefore also disclosed are methods of screening for an agent thatinhibits a T-cell mediated inflammatory response, wherein theinflammatory response comprises a response to a viral antigen, andwherein the viral antigen is selected from the group consisting ofHerpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus,Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variolavirus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus,Coronavirus, Influenza virus A, Influenza virus B, Measles virus,Polyomavirus, Human Papillomavirus, Respiratory syncytial virus,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus,Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebolavirus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitisvirus, Japanese Encephalitis virus, St. Louis Encephalitis virus, MurrayValley fever virus, West Nile virus, Rift Valley fever virus, RotavirusA, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiencyvirus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus,Simian immunodeficiency virus, Human Immunodeficiency virus type-1, andHuman Immunodeficiency virus type-2. Each and any of these viralantigens may also be excluded.

52. Also disclosed are methods of screening for an agent that inhibits aT-cell mediated inflammatory response, wherein the inflammatory responsecomprises a response to a bacterial antigen. For example, the bacteriumChlamydia pneumonia has been implicated in the pathogenesis of multiplesclerosis. Thus specifically disclosed are methods of screening for anagent that inhibits a T-cell mediated inflammatory response, wherein theinflammatory response comprises a response to Chlamydia pneumonia.Additionally, Nueroborrelisis (Lyme's disease of the central nervoussystem) can resemble MS. Thus specifically disclosed are methods ofscreening for an agent that inhibits a T-cell mediated inflammatoryresponse, wherein the inflammatory response comprises a response toBorrelia burgdorferi (the bacterium that causes Lyme's disease).

53. Therefore also disclosed are methods of screening for an agent thatinhibits a T-cell mediated inflammatory response, wherein theinflammatory response comprises a response to a bacterial antigen, andwherein the bacterial antigen is selected from the group consisting ofM. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M.avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M.ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides,other Nocardia species, Legionella pneumophila, other Legionellaspecies, Salmonella typhi, other Salmonella species, Shigella species,Yersinia pestis, Borrelia burgdorferi, Pasteurella haemolytica,Pasteurella multocida, other Pasteurella species, Actinobacilluspleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucellaabortus, other Brucella species, Cowdria ruminantium, Chlamydiapneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Chlamydiapneumonia, Coxiella burnetti, other Rickettsial species, Ehrlichiaspecies, Staphylococcus aureus, Staphylococcus epidermidis,Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis,Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserriameningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, otherPseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, otherHemophilus species, Clostridium tetani, other Clostridium species,Yersinia enterolitica, and other Yersinia species. Each and any of thesebacterial antigens may also be excluded.

54. Cancer, and in particular cancer antigens, are known to induce aninflammatory response in a subject. Thus also disclosed are methods ofscreening for an agent that inhibits a T-cell mediated inflammatoryresponse, wherein the inflammatory response comprises a response to acancer antigen. Cancer antigens are well known in the art and therefore,specifically disclosed are methods, wherein the antigen is related to acancer selected from the group consisting of lymphomas (Hodgkins andnon-Hodgkins), B cell lymphoma, T-cell lymphoma, myeloid leukemia,leukemias, mycosis fungoides, carcinomas, carcinomas of solid tissues,squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, blastomas,neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas,hypoxic tumours, myelomas, AIDS-related lymphomas or sarcomas,metastatic cancers, bladder cancer, brain cancer, nervous system cancer,squamous cell carcinoma of head and neck, neuroblastoma/glioblastoma,ovarian cancer, skin cancer, liver cancer, melanoma, squamous cellcarcinomas of the mouth, throat, larynx, and lung, colon cancer,cervical cancer, cervical carcinoma, breast cancer, epithelial cancer,renal cancer, genitourinary cancer, pulmonary cancer, esophagealcarcinoma, head and neck carcinoma, hematopoietic cancers, testicularcancer, colorectal cancers, prostatic cancer, or pancreatic cancer. Eachand any of these cancer antigens may also be excluded. In addition, eachand any inflammatory condition arising from cancer (i.e., malignancy)may be excluded.

55. It is understood that the disclosed screening methods can be used inexperimental settings. Such settings can require the induction of theT-cell mediated inflammatory response in order for an agent to haveinflammation available for inhibition. It is understood that thenecessity of inducing the inflammatory response is known to those ofskill in the art. That is, those of skill in the art will recognize ifthe inflammatory response being inhibited needs to be induced and howthe induction can occur. Thus, specifically contemplated are methods ofscreening for an agent that inhibits a T-cell mediated inflammatoryresponse in a subject, comprising the steps of a) administering theagent to the subject, b) inducing the inflammatory response in thesubject, and c) detecting CXCL13 in the subject, wherein a reduction inthe level of CXCL13 in the subject as compared to a control levelindicates an agent that inhibits an inflammatory response. Optionalllystep (a) can precede, follow, or occur simultaneously with step (b).

56. Many different inducers are known in the art and one of skill in theart will understand the appropriate inducer to use for the inflammatoryresponse being studied. It is understood that the inflammatory responsecan be induced by a peptide, polypeptide, or protein. For example, theinducer can be a myelin protein such as myelin basic protein. In the EAEmodel of MS the inflammatory condition can be induced by proteolipidprotein (PLP), myelin oligodendrocyte protein (MOG), myelin basicprotein (MBP) or an antigenic fragment thereof (e.g., PLP₁₃₅₋₁₅₅ andPLP₁₃₉₋₁₅₁ MBP_(Ax1-11) or MOG₃₅₋₅₅).

57. The disclosed methods can also be performed using other molecules toassess inhibition. For example, CXCR5, the ligand for CXCL13 may be usedto detect inhibition. Thus also disclosed are methods of screening foran agent that inhibits a T-cell mediated inflammatory response in asubject, comprising the steps of a) administering the agent to thesubject, b) inducing the inflammatory response in the subject, and c)detecting CXCR5 in a sample from the subject, a reduction in the levelof CXCR5 in the subject as compared to a control level indicating anagent that inhibits an inflammatory response.

58. The disclosed screening methods can use tissue samples from asubject to detect the presence of CXCL13 or CXCR5. Thus alsocontemplated are methods of screening for an agent that inhibits T-cellmediated inflammatory responses in a subject with an inflammatoryresponse comprising administering to the subject the agent and detectingthe presence of CXCL13 or CXCR5, wherein the CXCL13 or CXCR5 is detectedin a tissue sample from the subject.

59. The tissue samples can be solid tissue or organs as well as fluidtissue. Thus disclosed are screening methods wherein the tissue samplecomprises a blood, lymphoid tissue samples (e.g., lymph node, splenictissue, bone marrow), cerebrospinal fluid, synovial fluid. Tissuesamples may be taken from the site of inflammation or at other sites. Itis understood that the tissue sample can comprise lymphoid tissue, butcan also comprise non-lymphoid tissue.

60. The disclosed screening methods use CXCL13 or CXCR5 as markers toassess inhibition. The art is replete with examples of methods ofdetecting cellular markers. For example surface markers and theirligands can be detected using antibodies specific to the marker ofinterest. Therefore specifically disclosed methods of screening for anagent that inhibits a T-cell mediated inflammatory response in a subjectwith an inflammatory response comprising administering to the subjectthe agent, inducing the inflammatory response when necessary, anddetecting the presence of CXCL13 or CXCR5 in the subject, wherein CXCL13or CXCR5 is detected by staining the tissue sample with CXCL13 or CXCR5antibodies respectively, wherein the antibodies are linked to adetectable moiety. Assays used to detect antibodies are well-known inthe art and include but are not limited to flow cytometry,immunohistochemistry, ELISA, and ELISpot.

61. In some instances it can be necessary to determine the specificityand activation state of infiltrating T-cells that are detected due tothe presence of CXCR5 on their surface. The advantage of knowing theT-cell specificity and activation state is the ability to determine ifthe CXCR5+ T-cells are specific to the inflammation being inhibited ordirected to unrelated antigens. Thus, only the targeted T-cells areevaluated to assess inhibition. Therefore, specifically disclosed arescreening methods further comprising determining the antigen specificityof CXCR5 positive T-cells in the sample. Also disclosed are screeningmethods further comprising determining the activation state of any CXCR5positive T-cells in the sample. Many markers for assessing theactivation state are known in the art. Such markers can include but arenot limited to ICOS, CD11a, CD45RO, CD45RA, CD44, CD62L, CD27, andCD43).

Methods of Treatment

62. Agents, including but not limited to, those identified via thescreening methods disclosed herein can be used for the treatment ofT-cell mediated inflammation specifically providing a treatment forinflammatory conditions, including but not limited to inflammatoryconditions such as multiple sclerosis (MS). Thus, one embodiment of thedisclosed invention is a method of treating a subject with multiplesclerosis, comprising administering to the subject a therapeutic amountof an agent, e.g., an agent identified by the disclosed screeningmethods. For example, disclosed are methods of treating a subject withMS, comprising administering to the subject a therapeutic amount of theagent identified by the disclosed screening methods.

63. The disclosed compositions can be used to treat a subject marked byany disease with uncontrolled T-cell mediated inflammation. For example,specifically disclosed are methods of treating a subject with aninflammatory condition comprising administering to the subject aneffective amount of an agent that inhibits the interaction of CXCL13 anda receptor that specifically binds CXCL13 (e.g., CXCR5), wherein theinhibition of interaction of CXCL13 with a receoptor (e.g., CXCR5)reduces the inflammatory condition.

64. Reduction in the inflammatory condition is determined by assessing avariety of clinical and laboratory parameters. Such parameters includethe frequency and/or size of gadolinium-enhancing lesions detected bybrain or spinal cord MRI scans, white matter lesion burden determined byMRI scanning, cerebrospinal fluid pleocytosis, cerebrospinal fluid IgGsynthesis rate and/or IgG index, cerebrospinal fluid oligoclonalbanding, serum anti-myelin antibody titers, serum autoreactive antibodytiters, the frequency of autoreactive T-cells among peripheralmononuclear cells (including myelin-specific T-cells), C-reactiveprotein, erythrocyte sedimentation rate and serum biomarkers orsurrogate markers that have yet to be defined.

65. The disclosed treatment methods employ agents to inhibitinflammation, prevent inflammation (i.e., prophylactically), or reduceongoing inflammation. The agents can include, but are not limited toantibodies that bind CXCL13. These antibodies include neutralizingantibodies that can prevent CXCL13 from binding to its receptor(s),e.g., CXCR5 and/or CXCR3 (i.e., a blocking antibody), or otherwiseinterfere with its biological activity. It is understood that theantibody can be a polyclonal or monoclonal antibody or antigenicfragments thereof. The antibody can be a naturally-occurring antibody.The antibody can also be an engineered form of antibody, including butnot limited to, a single chain antibody, a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, a scFv fragment (i.e., single chainvariable region), a minibody, a dimeric antibody (diabody), a trimericantibody (triabody), or a tetrameric antibody (tetrabody). The antibodyor antibody fragment can be used as a fusion protein. In one embodiment,the agent is a human monoclonal antibody or fragment thereof thatspecifically binds CXCL13 and has neutralizing activity as determined byany suitable assay disclosed herein or known in the art. The agent canalso comprise a soluble form of CXCR5 to compete with the membrane boundligand and limit the signaling ability of the chemokine CXCL13.Likewise, the agent can comprise a soluble form of any other receptor(e.g., CXCR3) to which CXCL13 binds and through which CXCL13 exertsbiological activity as determined by an suitable assay disclosed hereinor known in the art, wherein the soluble receptor form competes withmembrane bound ligand and limits the signalling ability of CXCL13.

66. Thus, specifically disclosed are methods of reducing theexacerbation of an inflammatory condition in a subject comprisingadministering to the subject an effective amount of an agent thatinhibits the interaction of CXCL13 and CXCR5, the inhibition of CXCL13interaction with CXCR5 reducing the exacerbation. The interactionbetween CXCL13 and CXCR5 provides the signaling through which T-cellsare drawn to an area of inflammation and stimulated to produceinflammatory cytokines. Thus any agent that blocks this interaction canbe used in the present methods. For example, specifically disclosed andherein contemplate are methods of treating a subject with aninflammatory condition comprising administering to the subject aneffective amount of an agent that inhibits the interaction of CXCL13 andCXCR5, wherein the agent is an antibody to CXCR5 and wherein theantibody blocks CXCL13 binding without causing signaling through CXCR5.The agent can also comprise a small organic molecule or a macromoleculethat binds to either CXCL13 or CXCR5 so as to inhibit interactionstherebetween. The agent can also comprise an antibody, small organicmolecule, or macromolecule that interferes with the interaction ofCXCL13 and any other receptor (e.g., CXCR3) or ligand to which CXCL13binds, and which inhibits and inflammatory response.

67. Therefore an embodiment of the present invention is a method ofinhibiting an inflammatory response in a subject comprisingadministering to the subject an effective amount of an agent thatinhibits the interaction of CXCL13 and CXCR5, the inhibition of CXCL13interaction with CXCR5 inhibiting the inflammatory response.

68. Also disclosed are methods of reducing a T-cell-mediatedinflammatory response or condition e.g., an autoimmune condition, in asubject, comprising administering to the subject in need thereof aneffective amount of an agent that inhibits CXCL13 activity, theinhibition of CXCL13 reducing the inflammatory response or condition.

69. The disclosed treatment methods can be used to treat inflammatoryconditions. Inflammatory conditions can include but are not limited toautoimmune conditions and generalized conditions marked by systemic orlocalized inflammation. Thus it is understood that disclosed are methodsof treating an inflammatory response or condition, wherein theinflammatory response or condition is selected from the group consistingof asthma, alopecia greata, systemic lupus erythematosus, rheumatoidarthritis, reactive arthritis, spondylarthritis, systemic vasculitis,insulin dependent diabetes mellitus, multiple sclerosis, experimentalallergic encephalomyelitis, Sjogren's syndrome, graft versus hostdisease, inflammatory bowel disease including Crohn's disease,ulcerative colitis, ischemia reperfusion injury, myocardial infarction,Alzheimer's disease, transplant rejection (allogeneic and xenogeneic),thermal trauma, any immune complex-induced inflammation,glomerulonephritis, myasthenia gravis, cerebral lupus, Guillain-Barresyndrome, vasculitis, systemic sclerosis, anaphlaxis, catheterreactions, atheroma, infertility, thyroiditis, ARDS, post-bypasssyndrome, hemodialysis, juvenile rheumatoid, Behcets syndrome, hemolyticanemia, pemphigus, bullous pemphigoid, stroke, atherosclerosis,scleroderma, psoriasis, sarcoidosis, transverse myelitis, acutedisseminated encephalomyelitis, post-infectious encephalomyelitis,subacute sclerosing panencephalitis, polymyositis, dermatomyositis,incusion body myopathy, and chronic inflammatory demyelinatingpolyradiculopathy. Each and any of these inflammatory responses orconditions may also be excluded. Thus, for example, in certainembodiments, the inflammatory condition is not asthma, not SLE, notrheumatoid arthritis, not myasthenia gravis, not diabetes (e.g., notinsulin dependent diabetes mellitus), or not transplant rejection. Eachand any inflammatory condition associated with cancer (i.e., malignancy)may be excluded, as well.

70. It is understood that inflammatory conditions can also includeneuroinflammatory responses or conditions including but not limited tochronic neuroinflammatory conditions, relapsing and remittingconditions, and chronic demyelinating conditions. For example, thedisclosed methods of treatment can be used to treat a neuroinflammatorycondition, wherein the neuroinflammatory condition is selected from thegroup consisting of multiple sclerosis (MS), experimental allergicencephalomyelitis (EAE), Guillain-Barre syndrome, Alzheimer's disease,transverse myelitis, acute disseminated encephalomyelitis,post-infectious encephalomyelitis, subacute sclerosing panencephalitis,and chronic inflammatory demyelinating polyradiculopathy. Thus, forexample, disclosed herein are methods of treating a neuroinflammatorycondition wherein the neuroinflammatory condition is multiple sclerosis.Each and any of these neuroinflammatory conditions may also be excluded.

71. The inflammatory conditions of the invention involve theinflammatory response of leukocytes. It is understood that CXCR5+ Bcells, T-cells, and NK T-cells can be involved in generating theinflammatory response and are recruited to the site of inflammation.Thus it is understood and disclosed herein that the recruited leukocytescan be B cells (including but not limited to memory B cells), T-cells(e.g., CD4 and CD8 T-cells) and NK T-cells. Lymphoid inducer cells, aunique subpopulation of hematopoetic cells, also express CXCR5 and canbe involved in the initiation or perpetuation of an inflammatorycondition. The inflammatory conditions treated by the disclosed methodscan be T-cell mediated. In particular, the T-cell mediated condition orresponse can be mediated by antigen-specific pathogenic CD4+ T-cells.More specifically, the disclosed T-cells can be myelin reactive T-cells.

72. As used herein, the inflammatory response or condition can beinduced by autoantibodies. Autoimmune diseases, for example, arecharacterized by such autoantibodies.

73. The CXCL13 affected by the methods of the invention are produced byCD11c+ myeloid dendritic cells, e.g., in lesions of the central nervoussystem of the subject.

74. The agent optionally inhibits the action of CXCL13 in recruiting,positioning, and/or modulating CXCR5-expressing peripheral leukocytes orlymphoid tissue inducer cells in lesions. Furthermore, the agentoptionally inhibits the action of CXCL13 in recruiting, positioning,and/or modulating CXCR3-expressing B and/or T-cells in lesions. Thus theagent plays a significant role in interrupting the pathways involved inthe underlying inflammatory condition.

75. The leukocytes recruited, spatially positioned (with respect toother cell types), and/or modulated by the CXCL13 are effector/memory T(T_(em)) lymphocytes. The lymphocytes optionally have been activated byantigen and express iCOS, IL-7 receptor (IL-7R) and CD40 ligand (CD40L).Optionally, the recruited leukocytes are non polarized memory T-cells,myeloid cells, or CD4+ CD3-lymphoid tissue inducer cells.

76. The agent can inhibit the action of CXCL13 in facilitatinginteractions between CXCR5+ leukocytes within tissue infiltrates or caninhibit the action of CXCL13 in triggering lymphoid neogenesis. As usedherein, “lymphoid neogenesis” refers to development of organizedlymphoid structures within a target organ.

77. The agent can be an antisense oligonucleotide or an siRNA moleculethat inhibits expression of CXCL13. Such an antisense oligonucleotide orsiRNA should be sufficiently complementary to the sequence of DNA ormRNA encoding CXCL13 so as to have the proper antisenseor interferingproperty. The term “siRNA” as used herein refers to any nucleic acidmolecule capable of mediating RNA interference (“RNAi”) or genesilencing; see for example, Kreutzer et al., International PCTPublication No. WO 00/44895; Zemicka-Goetz et al., International PCTPublication No. WO 01/36646; Fire, International PCT Publication No. WO99/32619; Plaetinck et al., International PCT Publication No. WO00/01846; Mello and Fire, International PCT Publication No. WO 01/29058;Deschamps-Depaillette, International PCT Publication No. WO 99/07409;and Li et al., International PCT Publication No. WO 00/44914. Forexample, the siRNA can be a double-stranded polynucleotide moleculecomprising self-complementary sense and antisense regions, wherein theantisense region comprises complementarity to a target nucleic acidmolecule. The siRNA can be a single-stranded hairpin polynucleotidehaving self-complementary sense and antisense regions, wherein theantisense region comprises complementarity to a target nucleic acidmolecule. The siRNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises complementarity to a target nucleic acid molecule, andwherein the circular polynucleotide can be processed either in vivo orin vitro to generate an active siRNA capable of mediating RNAi. As usedherein, siRNA molecules need not be limited to those moleculescontaining only RNA, but further encompasses chemically-modifiednucleotides and non-nucleotides. In certain embodiments, the shortinterfering nucleic acid molecules of the invention lack 2′-hydroxy(2′-OH) containing nucleotides. Applicant describes in certainembodiments short interfering nucleic acids that do not require thepresence of nucleotides having a 2′-hydroxy group for mediating RNAi andas such, short interfering nucleic acid molecules of the inventionoptionally do not contain any ribonucleotides (e.g., nucleotides havinga 2′-OH group). The modified short interfering nucleic acid molecules ofthe invention can also be referred to as short interfering modifiedoligonucleotides “siMON.” As used herein, the term siRNA is meant to beequivalent to other terms used to describe nucleic acid molecules thatare capable of mediating sequence specific RNAi, for example shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA, shorthairpin RNA (shRNA), short interfering oligonucleotide, shortinterfering nucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing.

78. In one embodiment of the present invention, each sequence of ansiRNA molecule of the invention is independently 18 to 24 nucleotides inlength, in specific embodiments about 18, 19, 20, 21, 22, 23, or 24nucleotides in length. In another embodiment, the siNA duplexes of theinvention independently comprise between about 17 and about 23 basepairs. In yet another embodiment, siRNA molecules of the inventioncomprising hairpin or circular structures are about 35 to about 55nucleotides in length, or about 38 to about 44 nucleotides in length andcomprising 16-22 base pairs.

79. This invention provides a method of inhibiting extralymphaticlymphoid neogenesis in a subject comprising administering to the subjectan effective amount of an agent that inhibits CXCL13, the inhibition ofCXCL13 inhibiting lymphoid neogenesis.

80. Further provided are methods of inhibiting accumulation and/orstimulation of T_(em) cells or activated B cells in a CNS inflammatorysite in a subject, comprising administering to the subject an effectiveamount of an agent that inhibits CXCL13, CXCL13 inhibition therebyinhibits accumulation and/or stimulation through the CXCR5 receptor.Also disclosed are methods of inhibiting B cell activation ordifferentiation in a CNS inflammatory site in a subject, comprisingadministering to the subject an agent that inhibits CXCL13, inhibitionof CXCL13 inhibiting B cell activation or differentiation. Alsodisclosed are methods of inhibiting the migration of CD4+CXCR5+ T_(em)cells or CXCR5+ B cells from the periphery into the CNS in a subject,comprising administering to the subject an effective amount an agentthat inhibits the action of CXCL13 on said T or B cells, inhibition ofCXCL13 inhibiting said migration.

81. Inflammation can occur anywhere in a subject. Therefore it isnecessary for the agents to be administered systemically in the event ofsystemic inflammatory conditions or administered locally or regionally,in the event of local or regional inflammatory conditions. It isunderstood that one of skill in the art will be able to determine if theinflammation is systemic or local and can administer the agentaccordingly.

82. It is well known in the art that some inflammatory conditions arechronic in nature. Moreover, it is understood that some chronicinflammatory conditions can appear to be under control, but re-emerge orrelapse. Thus specifically contemplated are methods of treating a T-cellmediated inflammatory response or condition in a subject, comprisingadministering to the subject in need thereof an effective amount of anagent that inhibits CXCL13, wherein the agent is administered after theinflammatory response or condition has been initially induced but beforea first relapse. Also disclosed are methods of treatment of a T-cellmediated inflammatory response or condition, wherein the agent isadministered at the time of a first relapse. Also disclosed are methodsof treatment of a T-cell mediated inflammatory response or condition,wherein the agent is administered at the time of, or following, theinitial clinical exacerbation (ie, the presenting episode) but prior toa first clinical relapse. Also disclosed are methods of treatment of aT-cell mediated inflammatory response or condition, wherein the agent isadministered when subclinical inflammatory activity has been detected(ex. by MRI scans or blood tests) that is likely to evolve into aclinical syndrome in the future. Also disclosed are methods of treatmentof a T-cell mediated inflammatory response or condition, wherein theagent is administered after a first relapse. For example, specificallycontemplated are methods of treatment of the invention, wherein saidadministration is performed at the time of relapse of a chronicneuroinflammatory condition. Also disclosed are method of inhibiting thebinding or other interactions between CXCL13 and a CXCR5-expressing cell(or a cell expressing any other receptor that interacts with CXCL13,e.g., CXCR3) that can participate in the induction, progression orexpression of a T-cell-mediated neuroinflammatory response, comprisingproviding to said cell an amount of an agent effective in inhibitingCXCL13 binding to said cell. In one embodiment, the neuroinflammatoryresponse is multiple sclerosis.

83. It is disclosed and herein contemplated that administration of anagent to treat an inflammatory condition may not be curative, but mayreduce the inflammation. Such an agent would then be needed for the lifeof the subject or until the inflammatory condition is eliminated. Thusalso disclosed are methods of the invention, wherein the agent isadministered chronically. Also disclosed are methods of the invention,where the administration of said agent aborts the relapse, or results inmore complete and or more rapid recovery from a first or subsequentrelapse. It is also understood and herein contemplated thatadministration of the disclosed agents can halt the progression of achronic inflammatory condition. It is also understood that suchtreatment can prevent further episodes of an inflammatory condition.Thus also disclosed are method of the invention, wherein theadministration of said agent stabilizes the clinical status of a patientwith a chronic inflammatory condition. (prevents or reduces futureaccumulation of deficits). Such long term administrations are well-knownin the art and can involve daily, weekly, or monthly adminstrations ofthe agent or alternatively the agent can be administered in acontrolled-release or depot formulation.

84. It is understood that inflammatory conditions can have multipleeffects on a subject which result in undersireable symptoms. It is alsounderstood that there are circumstances in which multiple agents will bepreferred to single agent administration for the control of inflammatoryconditions. Thus specifically disclosed are methods of treating aninflammatory condition wherein the agents of the treatment methodsdisclosed herein may be administered in combination with one or moreadditional drugs that are useful for (a) inhibiting the inflammatoryresponse or condition, and/or (b) treating any other undesiredassociated symptom. It is recognized that one of skill in the art willbe able to determine if combination therapy is preferred over singleagent use.

Antibodies

Antibodies Generally

85. The terms “antibody,” “antibodies,” “immunoglobulins,” or“immunoglobulins” refer to antibodies comprised of two immunoglobulinheavy chains and two immunoglobulin light chains as well as a variety offorms besides antibodies; including, for example, Fv, Fab, and F(ab′)2as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al.,Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et al.,Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al.,Science 242, 423-426 (1988), which are incorporated herein byreference). (See, generally, Hood et al., Immunology, Benjamin, N.Y.,2ND ed. (1984), Harlow and Lane, ANTIBODIES. A LABORATORY MANUAL, ColdSpring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature, 323,15-16 (1986), which are entirely incorporated herein by reference).

86. The antibodies for use in the present invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized orchimeric antibodies, single chain antibodies, scFv fragments, Fabfragments, F(ab′)2 fragments, fragments produced by a Fab expressionlibrary, domain-deleted antibodies (including, e.g., CH2 domain-deletedantibodies), anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id antibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. The term “antibody,” as used herein,refers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule.

87. The antibodies for use in the present invention also include, butare not limited to, engineered forms of antibodies and antibodyfragments such as diabodies, triabodies, tetrabodies, and highermultimers of scFvs, as well as minibodies, such as two scFv fragmentsjoined by two constant (C) domains. See, e.g., Hudson, P. J. andCouriau, C., Nature Med. 9: 129-134 (2003); U.S. Publication No.20030148409; U.S. Pat. No. 5,837,242 (all of which are entirelyincorporated by reference herein).

88. The antibodies for use in the present invention may be from anyanimal origin including birds and mammals. Preferably, the antibodiesare human, murine (e.g., mouse and/or rat), donkey, ship rabbit, goat,guinea pig, camel, horse, or chicken. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulin and that do not express endogenous immunoglobulins, asdescribed, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati etal. The antibodies are tested for their desired activity using the invitro assays described herein, or by analogous methods, after whichtheir in vivo therapeutic and/or prophylactic activities are testedaccording to known clinical testing methods.

89. The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855, 1984, all of which are herein incorporatedby reference in their entireties).

90. Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies of the invention can be prepared using hybridomamethods, including those known in the art such as those described byKohler and Milstein, Nature, 256:495, 1975; Harlow et al., ANTIBODIES: ALABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties). In a hybridoma method, a mouse or otherappropriate host animal is typically immunized with an immunizing agentto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the immunizing agent.Alternatively, the lymphocytes may be immunized in vitro.

91. The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced. For example, the monoclonal antibodiesmay also be made by recombinant DNA methods, such as those described inU.S. Pat. No. 4,816,567 (Cabilly et al.) (herein incorporated byreference in its entirety). DNA encoding the monoclonal antibodies ofthe invention can be readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). Libraries of antibodies or active antibody fragmentscan also be generated and screened using phage display techniques, e.g.,as described in U.S. Pat. No. 5,804,440 to Burton et al. and U.S. Pat.No. 6,096,441 to Barbas et al. (herein incorporated by reference intheir entireties).

92. In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art. For instance, digestion can be performedusing papain. Examples of papain digestion are described in WO 94/29348published Dec. 22, 1994 and U.S. Pat. No. 4,342,566 (herein incorporatedby reference in their entireties). Papain digestion of antibodiestypically produces two identical antigen binding fragments, called Fabfragments, each with a single antigen binding site, and a residual Fcfragment. Pepsin treatment yields a fragment that has two antigencombining sites and is still capable of cross-linking antigen.

93. The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr OpinBiotechnol 3:348-354, 1992). Examples of techniques which can be used toproduce single-chain Fvs and antibodies, as well as diabodies,triabodies, and tetrabodies, include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88(1991); Shu et al., PNAS 90:7995 -7999 (1993); Skerra et al., Science240:1038-1040 (1988); U.S. Application Publication No. 20020018749 andU.S. Pat. No. 5,837,242 (herein incorporated by reference in theirentireties).

94. Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

95. As used herein, the term “antibody” or “antibodies” can also referto a human antibody and/or a humanized antibody. Many non-humanantibodies (e.g., those derived from mice, rats, or rabbits) arenaturally antigenic in humans, and thus can give rise to undesirableimmune responses when administered to humans. Therefore, the use ofhuman or humanized antibodies in the methods of the invention serves tolessen the chance that an antibody administered to a human will evoke anundesirable immune response.

Human Antibodies

96. The human antibodies of the invention can be prepared using anytechnique. Examples of techniques for human monoclonal antibodyproduction include those described by Cole et al. (Monoclonal Antibodiesand Cancer Therapy, Alan R., Ed. Liss, p. 77, 1985) and by Boemer et al.(J Immunol, 147(1):86-95, 1991). Human antibodies of the invention (andfragments thereof) can also be produced using phage display libraries(Hoogenboom et al., J Mol Biol, 227:381, 1991; Marks et al., J Mol Biol,222:581, 1991).

97. For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library. Phage expressing an antigen bindingdomain that binds the antigen of interest can be selected or identifiedwith antigen, e.g., using labeled antigen or antigen bound or capturedto a solid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

98. As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science240:1041-1043 (1988) (all incorporated by reference in theirentireties).

99. Fully human antibodies can be produced according to the methodtaught by US 2002 0123057A1, “In vitro methods of producing andidentifying immunoglobulin molecules in eukaryotic cells,” published 5Sep. 2002, which is incorporated herein by reference in its entirety.Briefly, a mouse (or human) heavy chain V region (VH) linked to a humanheavy chain constant region (CH) is employed to select fully humanimmunoglobulin light chains from a library of such light chains thatwhen paired with the mouse (or human) VH confers specificity for thedesired antigen (e.g., CXCL13). The selected fully human immunoglobulinlight chains are then employed to select fully human immunoglobulinheavy chains from a library of such heavy chains that, when paired withthe fully human light chain, confer specificity for the desired antigen(e.g., CXCL13). Similarly, the mouse (or human) light chain V region(VL) linked to a human light chain constant region (CL) may be employedto select fully human immunoglobulin heavy chains from a library of suchheavy chains that when paired with the mouse (or human) VL confersspecificity for the desired antigen (e.g., CXCL13). The selected fullyhuman immunoglobulin heavy chains are then employed to select fullyhuman immunoglobulin light chains from a library of such light chainsthat, when paired with the fully human heavy chain, confer specificityfor the desired antigen (e.g., CXCL13). Frequently, the fully humanantibody selected in this fashion has epitope specificity that isidentical or closely related to that of the original mouse (or human)antibody.

100. The method of US Patent Application Publication No. 2002 0123057 μlmay also be used with a library of heavy or light chains of which allmembers have one or more non-human (e.g., murine) CDRs. In one example,each member of the library comprises a CDR3 region derived from anisolated murine monoclonal antibody specific for the desired antigen(e.g., CXCL13).

101. The human antibodies of the invention can also be obtained fromtransgenic animals. For example, transgenic, mutant mice that arecapable of producing a full repertoire of human antibodies, in responseto immunization, have been described (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551-255, 1993; Jakobovits et al.,Nature, 362:255-258, 1993; Bruggermann et al., Year in Immunol. 7:33,1993). Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. For example, the human heavy and light chainimmunoglobulin gene complexes may be introduced randomly or byhomologous recombination into mouse embryonic stem cells. Alternatively,the human variable region, constant region, and diversity region may beintroduced into mouse embryonic stem cells in addition to the humanheavy and light chain genes. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Monoclonal antibodies directed against theantigen can be obtained from the immunized, transgenic mice usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.13:65-93 (1995). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

102. Completely human antibodies which recognize a selected epitope canbe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Humanized Antibodies

103. Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFc, Fv, Fab, Fab′, or other antigen-binding portion of an antibody)which contains a portion of an antigen binding site from a non-human(donor) antibody integrated into the framework of a human (recipient)antibody.

104. To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525, 1986, Reichmann et al., Nature, 332:323-327, 1988, andPresta, Curr Opin Struct Biol, 2:593-596, 1992).

105. Methods for humanizing non-human antibodies are well known in theart. For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525,1986, Riechmann et al., Nature, 332:323-327, 1988, Verhoeyen et al.,Science, 239:1534-1536, 1988), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

Administration of Antibodies

106. Antibodies of the invention are preferably administered to asubject in a pharmaceutically acceptable carrier. Suitable carriers andtheir formulations are described in Remington: The Science and Practiceof Pharmacy (19th ed.) A. R. Gennaro, Ed., Mack Publishing Company,Easton, Pa. 1995. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarrier include, but are not limited to, saline, Ringer's solution anddextrose solution. The pH of the solution is preferably from about 5 toabout 8, and more preferably from about 7 to about 7.5. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped particles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

107. The antibodies can be administered to the subject, organ, tissue,or cell by a variety of methods. For example, the antibody can be addedto in vitro culture. Various other delivery systems are known and can beused to administer a compound of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the compound, construction of a nucleic acid as part of aretroviral or other vector, etc. Methods of introduction include but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The compounds orcompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compounds or compositions ofthe invention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. Local orintravenous injection is preferred.

108. In one embodiment, the entire antibody dose is provided in a singlebolus. Alternatively, the dose can be provided by multipleadministrations, such as an extended infusion method or by repeatedinjections administered over a span of hours or days.

109. In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

110. In another embodiment, the compound or composition can be deliveredin a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in LIPOSOMES IN THE THERAPY OFINFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.).

111. In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see MEDICAL APPLICATIONS OF CONTROLLED RELEASE, LANGER AND WISE(eds.), CRC Pres., Boca Raton, Fla. (1974); CONTROLLED DRUGBIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE,supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems arediscussed in the review by Langer (Science 249:1527-1533 (1990)).

112. Effective dosages and schedules for administering the antibodiesmay be determined empirically, and making such determinations is withinthe skill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. Guidance in selecting appropriate doses forantibodies is found in the literature on therapeutic uses of antibodies,e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., NogesPublications, Park Ridge, N.J., 1985 ch. 22 and pp. 303-357; Smith etal., Antibodies in Human Diagnosis and Therapy, Haber et al., eds.,Raven Press, New York, 1977 pp. 365-389. A typical daily dosage of theantibody used alone might range from about 1 μg/kg to up to 100 mg/kg ofbody weight or more per day, depending on the factors mentioned above.

Pharmaceutical Carriers/Delivery of Pharmaceutical Products

113. As described above, the compositions can also be administered invivo in a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, i.e., the material may be administered to a subject, alongwith the cell, without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical composition in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

114. The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, intranasally, topically orthe like. The exact amount of the compositions required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the severity of the disorder being treated,the particular cell used, its mode of administration and the like. Thus,it is not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

115. Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

116. The materials may be in solution, suspension (for example,incorporated into microparticles, liposomes, or cells). These may betargeted to a particular cell type via antibodies, receptors, orreceptor ligands. The following references are examples of the use ofthis technology to target specific proteins to tumor tissue (Senter, etal., Bioconjugate Chem, 2:447-451, 1991; Bagshawe, K. D., Br J Cancer,60:275-281, 1989; Bagshawe, et al., Br J Cancer, 58:700-703, 1988)Senter, et al., Bioconjugate Chem, 4:3-9, 1993; Battelli, et al., CancerImmunol Immunother, 35:421-425, 1992; Pietersz and McKenzie, Immunolog.Reviews, 129:57-80, 1992; and Roffler, et al., Biochem Pharmacol,42:2062-2065, 1991). Vehicles such as “stealth” and other antibodyconjugated liposomes (including lipid mediated drug targeting to coloniccarcinoma), receptor mediated targeting of DNA through cell specificligands, lymphocyte directed tumor targeting, and highly specifictherapeutic retroviral targeting of murine glioma cells in vivo. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Hughes et al., Cancer Res.,49:6214-6220, 1989; and Litzinger and Huang, Biochimica et BiophysicaActa, 1104:179-187, 1992). In general, receptors are involved inpathways of endocytosis, either constitutive or ligand induced. Thesereceptors cluster in clathrin-coated pits, enter the cell viaclathrin-coated vesicles, pass through an acidified endosome in whichthe receptors are sorted, and then either recycle to the cell surface,become stored intracellularly, or are degraded in lysosomes. Theinternalization pathways serve a variety of functions, such as nutrientuptake, removal of activated proteins, clearance of macromolecules,opportunistic entry of viruses and toxins, dissociation and degradationof ligand, and receptor-level regulation. Many receptors follow morethan one intracellular pathway, depending on the cell type, receptorconcentration, type of ligand, ligand valency, and ligand concentration.Molecular and cellular mechanisms of receptor-mediated endocytosis havebeen reviewed (Brown and Greene, DNA Cell Biol 10:6, 399-409, 1991).

Pharmaceutically Acceptable Carriers

117. The compositions, including antibodies, can be used therapeuticallyin combination with a pharmaceutically acceptable carrier.

118. Pharmaceutical carriers are known to those skilled in the art.These most typically would be standard carriers for administration ofdrugs to humans, including solutions such as sterile water, saline, andbuffered solutions at physiological pH. The compositions can beadministered intramuscularly or subcutaneously. Other compounds will beadministered according to standard procedures used by those skilled inthe art.

119. Pharmaceutical compositions may include carriers, thickeners,diluents, buffers, preservatives, surface active agents and the like inaddition to the molecule of choice. Pharmaceutical compositions may alsoinclude one or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

120. The pharmaceutical composition may be administered in a number ofways depending on whether local or systemic treatment is desired, and onthe area to be treated.

Administration may be topically (including ophthalmically, vaginally,rectally, intranasally), orally, by inhalation, or parenterally, forexample by intravenous drip, subcutaneous, intraperitoneal orintramuscular injection. The disclosed antibodies or agents can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally.

121. Preparations for parenteral administration include sterile aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

122. Formulations for topical administration may include ointments,lotions, creams, gels, drops, suppositories, sprays, liquids, andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners, and the like may be necessary or desirable.

123. Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

124. Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,tri-alkyl and aryl amines and substituted ethanolamines.

Therapeutic Uses

125. The dosage ranges for the administration of the compositions arethose large enough to produce the desired effect in which the symptomsdisorder are affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days. For antibodies, the dosage administered to apatient is typically 0.1 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between 0.1mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kgto 10 mg/kg of the patient's body weight. Generally, human antibodieshave a longer half-life within the human body than antibodies from otherspecies due to the immune response to the foreign polypeptides. Thus,lower dosages of human antibodies and less frequent administration isoften possible. Further, the dosage and frequency of administration ofantibodies of the invention may be reduced by enhancing uptake andtissue penetration (e.g., into the brain) of the antibodies bymodifications such as, for example, lipidation.

126. The invention also provides a pharmaceutical pack or kit comprisingone or more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

EXAMPLES

127. The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compounds, compositions, articles, devices and/or methodsclaimed herein are made and evaluated, and are intended to be purelyexemplary of the invention and are not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers (e.g., amounts, temperature,etc.), but some errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is in ° C.or is at ambient temperature, and pressure is at or near atmospheric.

Example 1

Methods

Mice.

128. Female SJL, B10.PL and C57BL/6 mice were obtained from JacksonLaboratories (Fredrick, Md.). CXCL13 deficient mice on the C57BL/6background were also obtained (Ansel, K. M., et al. 2000. Nature406:309-314, Ansel, K. M., et al. 2002. Immunity 16:67-76). All animalswere housed under specific-pathogenfree, barrier facility conditions.

Peptides.

129. Myelin peptides were synthesized by Macromolecular Resources (FortCollins, Colo.) and purified by HPLC. The sequences were as follows:Proteolipid protein (PLP)₁₃₉₋₁₅₁: HSLGKWLGHPDKF (SEQ ID NO: 1); Myelinbasic protein (MBP)_(Ac1-11): Ac-ASQKRPSQRHG (SEQ ID NO: 2) and myelinoligodendrocyte glycoprotein (MOG)₃₅₋₅₅: MEVGWYRSPFSRVVHLYRNGK (SEQ IDNO: 3).

Induction of EAE by Active Immunization.

130. Mice were immunized with 100 μg of the relevant myelin peptideemulsified in CFA (with 4 mg/ml heat-killed Mycobacteria tuberculosisH37Ra; vol:vol) by subcutaneous (s.c.) injection at four sites over theflanks. Bordetella pertussis toxin (List Laboratories) was injectedintraperitoneally (i.p., 200 ng/mouse) on days 0 and 2 post-challenge.Animals were examined daily for signs of EAE and rated for severity ofneurological impairment on a 5 point scale as previously described(Segal, B. M., and Shevach, E. M. 1996. J Exp Med 184:771-775).

Induction of EAE by Adoptive Transfer.

131. SJL mice were immunized with 100 μg of PLP₁₃₉₋₁₅₁ (SEQ ID NO: 1)emulsified in IFA (1:1) and C57BL/6 mice were immunized with MOG₃₅₋₅₅ inCFA (1:1) by the s.c. route as described above, but without injection ofpertussis toxin. Twelve to fourteen days later draining LNs (inguinaland axillary) were removed and processed as previously described (Segal,B. M., et al. 2000. J Immunol 164:5683-5688). Cells were cultured instandard media with myelin peptide (50 μg/ml PLP or 25 μg/ml MOG) withor without murine rIL-12 (5 ng/ml; R&D Systems). At 96 h cells wereharvested, washed and adoptively transferred into naïve syngeneicrecipients (40-50×10⁶ cells i.p.). Recipient mice were examined dailyand rated by observers blinded to treatment group or phenotype.

RNA Analysis.

132. Total RNA was extracted from whole spinal cords or isolatedmononuclear cells using Trizol reagent (GIBCO BRL). Multiprobe RNaseprotection assays (RPA) were performed with the mCK-5b template sets,Riboquant In Vitro Transcription and RPA Kits (PharMingen). Riboprobetemplates for CCL19, CCL21, and CXCL13 were obtained from Torrey PinesBiolabs, Inc. (Houston, Tex.); the template for Cyclophilin was fromAmbion. RPA products were resolved on a denaturing polyacrylamide geland quantified by Phosphorimaging. For RT-PCR, 2.5 μg of total RNA wasreverse transcribed using random hexamer primers and M-MLV reversetranscriptase (GIBCO-BRL). cDNA was amplified using the following primerpairs (forward, reverse, and product size): CXCL13, TGAGGCTCAGCACAGCAACG(SEQ ID NO: 4) and CTTGAGCATTCCCTCTCAGCT (SEQ ID NO: 5)(537 bp); CCL19,CTGCCTCAGATTATCTGCCAT (SEQ ID NO: 6) and GCCAGAGTGATTCACATCTCT (SEQ IDNO: 7) (371 bp); CXCR5, ATGAACTACCCACTAACCCTG (SEQ ID NO: 8) andAGGTGAACCAGGCTCTAGTTT (SEQ ID NO: 9) (658 bp); CCR7,GTGCTGGTGGTGGCTCTCCTTGTC (SEQ ID NO: 10) and CGTGTCCTCGCCGCTGTTCTTC (SEQID NO: 11) (594 bp); Class II transactivator form 1 (CIITA-I),GTGATGCCCTGGCCCGGAAGATTT (SEQ ID NO: 12) and TCGGGGAGACTGGGGATACTGAGG(SEQ ID NO: 13) (766 bp); CD38, GGGAGCCCCTTCAAGATACAAGTGACC (SEQ ID NO:14) and CGGGGCCAGTTCCCTCCAAGAC (SEQ ID NO: 15) (452 bp); mb-1,GCCAGGGGGTCTAGAAGC (SEQ ID NO: 16) and TCACTTGGCACCCAGTACAA (SEQ ID NO:17) (308 bp); Lysozyme M, CAGGCCAAGGTCTATGAACG (SEQ ID NO: 18) andATTGTATGGCTGCAGTGATGTC (SEQ ID NO: 19) (289 bp); HPRT,GTTGGATACAGGCCAGACTTTGTTG (SEQ ID NO: 20) and GAGGGTAGGCTGGCCTATAGGCT(SEQ ID NO: 21) (353 bp).

Western Blot Analysis.

133. The assay was performed following published protocols (Luther, S.A., et al. 2002. J Immunol 169:424-433). Briefly, spinal cord tissue waspooled from 10 mice and homogenized in ice-cold lysis buffer (0.1MTris-HCl (pH 8.0), 0.5 M EDTA, 60 mM CHAPS, Protease inhibitor cocktail(Sigma), at 1:50). Debris was removed by centrifugation (100,000 g for 1hour), and supernatant was immunoprecipitated using heparin-Sepharose.Detection was with goat anti-mouse CCL19, CCL21, CXCL13 antibodies (R&DSystems), followed by antigoat HRP (Jackson ImmunoResearchLaboratories). The bands were visualized using chemiluminescence (PIERCESuper Signal West Femto substrate). Positive controls included proteinextract from spleen, and recombinant chemokine proteins (R&D Systems).

Isolation of Spinal Mononuclear Cells.

134. Mice were anesthesized with Avertin and perfused with PBS by theintracardiac route using a peristaltic pump. Intact vertebral columnswere removed by gross dissection and cords were ejected under thepressure of an HBSS-filled syringe. Spinal cord tissue was then mincedinto small fragments and digested with collagenase (2 mg/ml; CLS-4,Worthington Biochemical Corporation) and DNAse (1 mg/ml; DN25, Sigma).Mononuclear cells (MNCs) were isolated over a 30%/70% Percoll gradient(Pharmacia Biotech AP, Uppsala, Sweden) using standard protocols.

Enrichment of CD11c+ cells.

135. CD11c+ cells were enriched from spinal cord MNCs using αFITC-coatedmagnetic beads following staining with FITC-conjugated anti-CD11cmonoclonal antibody (Miltenyi).

Flow Cytometric Analysis.

136. Spinal cord MNCs were incubated with “FcBlock” and stained withvarious combinations of fluorochrome-labeled antibodies to mouse CD4,CXCR5, CD11c, CD11b, IgM, iCOS, CD44, and CD62L or with isotype-matchedcontrols (Pharmingen). Cells were washed twice and fixed with 1%paraformaldehyde in PBS prior to analysis on a Becton-Dickinson FACSCalibur instrument with CellQuest software.

Histopathological Studies.

137. Spinal cords were dissected from mice following intracardiacperfusion with 4% paraformaldehyde. They were then decalcified inImmunocal and fixed in 10% buffered formalin. Paraffin-embedded sectionsof the cervical, thoracic and lumbar regions were stained with H&E,Trichrome or Luxol fast blue-periodic acid Schiff (LFB-PAS) for lightmicroscopy. To identify T-cells, selected sections were stained withmouse anti-human CD3; detection was with the EnVision+ System (allreagents from DAKO Cytomation). Astrocytes were labeled with rabbitanticow GFAP (DAKO) followed by biotinylated goat anti-rabbit IgG(Vector), Streptavidin-HRP and AEC (Jackson). Macrophages and activatedmicroglia were identified by biotinylated Ricinus Communis agglutinin(RCA) I (Vector).

Lymphoproliferation and Cytokine Secretion Assays.

138. LN cells (4×10⁵ in 0.2 ml) or splenocytes (2×10⁵ in 0.2 ml) werecultured with or without myelin peptide in quadruplicate for 4 days in96 well flat-bottom plates (Costar, Cambridge, Mass.). Wells were pulsedfor the final 16 hours of culture with 1 μCi [3H]TdR (NEN), andincorporated radioactivity was measured with a Betaplate scintillationcounter (Wallac, Gaithersburg). For cytokine measurements by ELISA,splenocytes were cultured in 24 well plates (5×10⁶ cells/2 ml/well) for72 hours. Supernatants were collected and analyzed using the OptEIAMouse IFNγ set (Pharmingen). ELISPOT assays were performed as previouslydescribed (Kawakami, N., et al. 2004. J Exp Med 199:185-197).

Results

CXCL13 is Expressed in the CNS During Acute EAE.

139. CXCL13 mRNA was detected in the target organs of autoantibodyassociated autoimmune diseases such as Sjogren's syndrome andexperimental systemic lupus erythematosis (Ishikawa, S., et al. 2001. JExp Med 193:1393-1402, (Salomonsson, S., et al. 2002. Scan J Immunol55:336-342). It was questioned whether the chemokine is also upregulatedin the CNS of animals with EAE, a prototypical Th1-mediated autoimmunedisease. Spinal cords were removed from B10.PL mice five weeks followingimmunization with MBP peptide in CFA. RNA was extracted from individualcords for RT-PCR analysis. CXCL13 mRNA was detected in every cord fromanimals with clinical EAE, but not in cords from naïve controls or fromone MBP-primed mouse that remained asymptomatic at the time of sacrifice(FIG. 1 a). CXCR5 mRNA was present in the CNS of some symptomatic mice.By comparison, mRNA encoding the lymphoid chemokine CCL19 and itsreceptor CCR7 was expressed at low levels in cords of naïve mice and athigher levels following disease onset. In synchrony with CXCL13, markersindicative of T-cell (CD3δ, dendritic cell (CIITA form I) and myeloidcell (lysozyme M) accumulation were detected.

Myelin-Reactive Lymph Node Cells Induce CXCL13 Expression in the CNS inthe Absence of Adjuvants.

140. Microbial products, such as heat killed M. tuberculosis in CFA, canmodulate the expression of cytokines in the CNS even when administeredsystemically. Although CXCL13 was not detected in the spinal cords ofcontrol mice immunized with PBS in CFA, whether myelin-specific T-cellsalone are sufficient to upregulate CXCL13 expression in the CNS wasdetermined.

141. It was previously shown that LN cells from SJL mice immunized withan emulsion of PLP₁₃₉₋₁₅₁ peptide in IFA (without Mycobacteria) transferEAE following in vitro stimulation with a combination of antigen andrecombinant IL-112. The same LN cells are not encephalitogenic if IL-12is omitted from the culture. Using this experimental system it was foundthat spinal cords from mice that had been injected with PLP/IFA primed,PLP/IL-12 reactivated cells 10-12 days earlier universally expressedCXCL13 and CXCR5 transcripts (FIG. 1B). By contrast, spinal cordsharvested on the same day from healthy control mice, injected with cellsthat had been reactivated with PLP only, failed to express CXCL13 andCXCR5. The appearance of CXCL13 in the CNS was associated withupregulation of the T-cell, B cell, and dendritic cell markers (CD3δ,mb-1, and CIITA form I, respectively).

CXCL13 is Expressed on the Protein, as Well as the mRNA, Level inInflamed Spinal Cords.

142. In order to determine whether CXCL13 mRNA correlates with proteinexpression Western blot analyses was performed on pooled spinal cordtissues from adoptive transfer recipients with EAE. CXCL13 protein wasreadily detectable in inflamed spinal cords but not in spinal cords fromnaïve controls (FIG. 1C). Similar results were obtained with regard toCCL19. On the other hand, CCL21 protein was constitutively expressed inthe CNS in the absence of inflammation, possibly reflecting a role ofthat chemokine in homeostatic lymphocyte trafficking through the CNS.

CNS CXCL13 Levels Rise Progressively During the Course of Relapsing EAE.

143. Having demonstrated that CXCL13 is expressed in the CNS duringacute EAE, CXCL13 expression was measured during the course ofrelapsing-remitting disease using RPA. SJL mice were actively immunizedwith PLP/CFA and representative animals were sacrificed during thepresenting episode, remission and relapse for spinal cord harvest andRNA extraction. As shown in FIG. 2 a, CXCL13 levels rose steadily overthe course of the disease. CXCL13 mRNA was not detectable in cords fromnaïve mice, as confirmed by RT-PCR and southern blot hybridization.CCL19 and CCL21 followed similar kinetics to CXCL13. By contrast, CNSexpression of the “inflammatory” chemokines MIP-1α, MIP-2 and RANTESwere highest during the initial exacerbation, fell during remission andincreased modestly during relapse (FIG. 2 b). These results indicatethat inflammatory chemokines are dominant during the first presentationof EAE, while CXCL13 is involved in the progression of chronic/relapsingdisease.

CXCR5+ cells accumulate in EAE infiltrates.

144. CXCL13 is the ligand for CXCR5, a receptor expressed by B cells,recently activated T-cells and follicular helper CD4+ T-cells (Kim, C.H., et al. 2001. J Exp Med. 193:1373-1381, Forster, R., et al. 1996.Cell 87:1037-1047, Ansel, K. M., et al. 1999. J Exp Med. 190:1123-1134).The RT-PCR data shown in FIG. 1 a, b indicated that CXCR5+ cellsinfiltrate the CNS in association with the induction of CXCL13. This wascorroborated by flow cytometric analyses of spinal cord MNCs. CXCR5+cells were readily detected in the inflamed CNS of afflicted miceirrespective of the method of EAE induction or the stage of disease atsacrifice. The vast majority of CXCR5+ cells were CD4+CD3+ T-cellsexpressing an effector memory phenotype (CD62Llo, CD44hi, iCOShi). Therelative percentage of CXCR5+ cells within the CD4+ T-cell compartmenttended to increase during successive stages of relapsing disease in SJLmice. Although IgM+ B cells, which universally express CXCR5, could befound among the CNS infiltrating cells, they were present in sparsenumbers.

145. Interestingly, a small population of CD4+CD3− cells in spinal cordmononuclear fractions (FIG. 3 a, right panel) was also consistentlydetected. The majority of these cells expressed IL-7Rα, and a percentageexpressed CXCR5. The cell surface phenotype CXCR5+IL-7Rα+CD4+ CD3− issuggestive of lymphoid tissue inducer cells, a unique subset ofhematopoetic cells implicated in the development of lymph nodes, Peyer'spatches and nasal-associated lymphoid tissue during embryogenesis(Luther, S. A., et al. 2003. J Exp Med. 197:1191-1198, Finke, D., et al.2002. Immunity 17:363-373).

CXCL13 mRNA is Detected in CD11c+ Spinal Cord Mononuclear Cells.

146. CXCL13 is produced by stromal cells in spleen and lymph nodes. Inaddition, CXCL13 is secreted by some hematopoetic cells. Thus, murineperitoneal macrophages constitutively express CXCL13 (Ansel, K. M., etal. 2002. Immunity 16:67-76). Furthermore, myeloid dendritic cellsisolated from the thymus and kidneys of mice with experimental lupuswere found to express CXCL13 (Ishikawa, S., et al. 2001. J Exp Med193:1393-1402).

147. In order to investigate whether hematopoetic cells are also thesource of CXCL13 in the CNS, spinal cord MNCs were isolated from micewith EAE for RT-PCR analysis. During acute EAE CNS-infiltrating cellsare comprised primarily of myeloid cells, including a significant subsetexpressing the dendritic cell marker, CD11c (FIG. 4 a). Lymphoid cells,most of which are CD4+ T-cells, are also present (FIG. 3 a). mRNAencoding CXCL13 was repeatedly detected, as well as CCL19 and CCL21, inthis mixed leukocyte population isolated from SJL mice during either thefirst or second episode of EAE (FIG. 4 a). Similar results were obtainedfrom MOG-sensitized C57BL/6 mice during their initial clinicalpresentation (FIG. 4 b). By contrast, CXCL13 was not detectable in MNCsisolated from the spinal cords of naïve mice, which are dominated byCD11blo cells (resident microglia) and lack a CD11c+ cell population.CXCL13 transcripts were enriched in MACS-isolated CD 11c+ cells (FIG. 4c), indicating that dendritic cells might be a major source of thechemokine in the inflamed CNS.

Actively Immunized CXCL13 Deficient Mice Experience a Relatively MildCourse of EAE and Do Not Experience Relapses.

148. In order to assess the physiological significance of CXCL13 in EAEC57BL/6 CXCL13−/− and wildtype mice were immunized with MOG peptide inCFA and monitored their clinical courses. CXCL13 deficient miceexhibited a significant decrease in disease severity during thepresenting episode, recovered more fully and were free of relapses bycomparison to their wildtype counterparts (FIG. 5 a, Table 1). TABLE ICourse of EAE induced by active immunization in CXCL13^(−/−) and WT miceCXCL13^(−/−) WT P^(a) Incidence 43/55 52/67 P = 1.0 Disease course: P<0.0001 monophasic 36 13 relapsing or chronic 7 39Summary of 4 experiments.^(a)Fisher's exact test two-sided P value.

149. During the first exacerbation, the degree of inflammation anddemyelination in individual mice correlated closely with the clinicalscore. In CXCL13−/− mice inflammatory infiltration and demyelinationwere restricted, in large part, to the subpial regions (FIG. 6 a, H&E).By contrast, wildtype mice exhibited more extensive involvement of allwhite matter tracts. Macrophages and activated microglia, identified bythe lectin RCA, were less numerous in white matter lesions of CXCL13−/−mice. On the other hand, there was no apparent difference in the degreeof CD3+ T-cell infiltration between the two groups (FIG. 6 a).

150. The difference in demyelination (loss of blue staining) andinflammatory cell infiltration between the groups was even morepronounced during later stages (day 32 after immunization), whenwildtype mice were in relapse, while CXCL13−/− mice remained inremission (FIG. 6 b, LFB-PAS). Furthermore, lesions in wildtype micewere characterized by a prominent degree of gliosis (indicated by GFAPstaining) and intrameningeal and subpial fibrosis (Trichrome), whereasCXCL13−/− mice showed only mild changes.

151. Flow cytometric analysis of spinal cord MNCs from symptomaticCXCL13−/− mice demonstrated a reduced percentage of CD11b+myeloid cellsby comparison to wildtype mice. This was most notable during therecovery phase following acute EAE (FIG. 7). A relative paucity ofCNS-infiltrating macrophages in CXCL13−/− mice might partially accountfor the milder white matter damage that these animals experience. Alower number of MNCs per spinal cord was consistently isolated fromCXCL13−/− than from wildtype mice, at all stages of disease (between20-60% lower, depending on stage). However, there was no majordifference in the composition of the lymphoid cell population, and alower percentage of CXCR5+cells in spinal cord MNCs from CXCL13−/−versus wildtype mice was not documented. Therefore, while the absolutenumber of CNS-infiltrating T-cells/cord was generally lower in CXCL13−/−mice, T-cells were not preferentially depleted.

Peripheral Myelin-Specific T-Cell Responses are Comparable in CXCL13Deficient and Wildtype Mice.

152. CXCL13 deficient mice exhibit impaired lymphoid organogenesis, witha paucity of Peyer's patches and most lymph nodes and disorganizedsplenic architecture (Ansel, K. M., et al. 2000. Nature 406:309-314,Luther, S. A., et al. 2003. J Exp Med. 197:1191-1198, Moser, B., andLoetscher, P. 2001. Nature Immunology 2:123-128, Cyster, J. G. 1999.Science 286:2098-2102). Although CXCL13−/− mice are not grosslyimmunodeficient, their relative resistance to induction of EAE couldpotentially be related to the failure of autoreactive T-cells to undergopriming in the periphery. To investigate that, myelin-specificproliferative and cytokine responses by splenocytes harvested fromCXCL13 deficient and wildtype mice were measured following activeimmunization with MOG peptide. Comparable frequencies of MOG-specificIL-2 producing cells were found in CXCL13−/− and wildtype mice.CXCL13−/− mice also mounted significant lymphoproliferative and IFNγresponses upon challenge with antigen in vitro (Table 2). Collectivelythis data indicates that MOG-specific CD4+ T-cells undergo priming,clonal expansion and differentiation in the absence of CXCL13. TABLE 2Splenic T-cell responses of sensitized CXCL13 knockout and wild typemice* CXCL13^(−/−) wt Proliferation No 3598 ± 348 3722 ± 1220 (cpm)stimulus MOG 40856 ± 5123 8792 ± 3417 p = 0.02 IFNγ No  0.36 ± 0.09 0.57± 0.13 production stimulus (ng/ml) MOG 16.21 ± 0.82 20.30 ± 3.44  p =0.06 Con A 23.49 ± 3.35 36.68 ± 1.32  p = 0.02 Frequency No 9 10 of IL-2stimulus producers MOG 94 ± 2 112 ± 9  p = 0.12 (per 5 × 105 cells)*MOG-specific CD4+ T-cell responses were measured by tritiated thymidineincorporation (proliferation), ELISA (IFNγ) and elispot (IL-2). Theresults shown represent an average of 3 mice/group +/− standarddeviation. Groups were compared using the Student's T-test.

CXCL13 Deficiency or Blockade Attenuates Adoptively Transferred EAE.

153. Based on the above results it was speculated that CXCL13 playsanon-redundant role during the effector stage of EAE, at a point beyondperipheral T-cell priming. Next, the clinical EAE was compared inchemokine deficient and sufficient mice following the adoptive transferof MOG/IL-12 stimulated LN cells from MOG/CFA primed, chemokinesufficient donors. CXCL13 recipients underwent a milder course of EAE,with delayed onset and accelerated and more complete recovery (FIG. 5b).

154. Immunological studies in knock-out mice are potentially subject toartifacts consequent to compensatory pathways that develop in responseto the life-long deficiency of a particular cytokine or chemokine.Therefore, the effects of CXCL13 neutralization on adoptivelytransferred EAE in immunocompetent SJL recipients were also tested. SJLmice were treated with a neutralizing goat anti-mouse CXCL13 antibody,isotype matched control antibodies or PBS on days 3, 6 and 10 post celltransfer. As shown in FIG. 5 c, the animals treated with anti-CXCL13were protected from EAE.

CXCL13 Deficient Mice can Generate Myelin-Reactive EncephalitogenicT-Cells that Initiate Cns Inflammation

155. CXCL13 deficient mice exhibit impaired lymphoid organogenesis, witha paucity of Peyer's patches, lymph nodes and disorganized splenicarchitecture. Although these mice are not grossly immunodeficient, itwas important to determine whether they could mount a myelin-specificT-cell response sufficient to initiate CNS inflammation.

156. Splenocytes harvested from CXCL13−/− mice immunized with MOG₃₅₋₅₅in CFA mounted significant proliferative, IL-2 and IFN-g responses uponchallenge with peptide in vitro (FIG. 12A). LN cells, though scarce inCXCL13−/− mice, also proliferated vigorously and secreted IFN-g in anantigen-specific manner. Spleens from immunized CXCL13−/− mice generallycontained a lower frequency of IL-2 and IFN-g producing cells (onaverage 2 to 3-fold) than WT spleens (FIG. 12A, lower panel, Exp. 2).Nevertheless, individual responses varied, with some CXCL13−/− miceshowing comparable cytokine production to WT mice (Exp. 1). MOG-specificmemory T-cells persisted in spleens of CXCL13−/− mice as evidenced byIFN-g and IL-2 recall responses measured as late as day 46 afterimmunization (Exp. 3).

157. Next the ability of CXCL13−/− mice to generate encephalitogenicT-cells was assessed in adoptive transfer experiments. Splenocytes fromMOG/CFA primed CXCL13−/− or WT mice were stimulated in vitro with MOGand IL-12 for 4 days, and then equal numbers of cells were injected intonaïve WT recipients. Donor cells from both groups induced a severe andpersistent form of EAE (FIG. 12B). However, recipients of CXCL13−/−effector cells underwent a slightly less aggressive course that was,nonetheless, statistically different from recipients of WT effectorcells.

Adoptively Transferred EAE is Attenuated in CXCL13−/− Mice

158. Based on the observations that CXCL13 is upregulated in the CNSduring EAE, it was speculated that it might play a distinct role duringthe effector stage of pathogenesis. Consequently, clinical EAE in CXCL13deficient and sufficient mice was compared following the adoptivetransfer of MOG specific CD4+ T-cells from WT donors (FIG. 5D). WTrecipients injected with 5×10⁶ CD4+ T-cells quickly progressed to severeEAE: 60% of mice died, and the surviving mice suffered conspicuouschronic deficits. By contrast, CXCL13−/− recipients underwent asignificantly milder course of EAE with delayed onset, reduced mortality(28%) and accelerated recovery. The majority of mice enjoyed complete ornear complete recovery. Similar results were obtained in two additionalexperiments using LN cells from primed WT donors.

Inflammatory CNS Infiltrates are Diminished in CXCL13−/− Mice,Particularly During Chronic Stages of EAE

159. In order to investigate whether CXCL13 deficiency had influencedthe recruitment and retention of leukocyte subsets in the CNS, MNCs fromWT and CXCL13−/− spinal cords were isolated during early and late stagesof EAE and were analyzed by flow cytometry. CXCL13−/− mice consistentlyyielded fewer mononuclear cells per spinal cord than WT mice (FIG. 13A).During the first exacerbation the cell yield from pooled CXCL13−/− cordswas 30% to 50% lower that from WT cords. The number of CNS MNCs felldramatically between the presenting episode of EAE and the subsequentremission in both groups. However, the decline was more pronounced inCXCL13−/− mice, which yielded 4 to 5 fold fewer spinal cord MNCs thantheir WT counterparts during remissions.

160. During acute EAE, there was no major difference in the subsetcomposition of infiltrating cells. In particular, the percentages ofCD4+ T-cells were comparable in CXCL13−/− and WT infiltrates (FIG. 13B),and these cells uniformly expressed CD44 and ICOS, indicative of anactivated state. Percentages of myeloid cells (includingCD45^(hi)CD11b^(hi) monocytes/macrophages and CD11 c+DCs) and CD4-CD3+cells (primarily CD8+ T-cells) were moderately reduced in CXCL13−/− CNSinfiltrates (FIGS. 13B and 14A).

161. The percentage of CD4+ T-cells among infiltrating leukocytesremained comparable between the groups at remission. Hence, the absolutenumber of infiltrating CD4+ T-cells was 4 to 5 fold lower in CXCL13−/−cords, in direct proportion to the relative reduction in total MNCs(FIG. 13A). On the other hand, monocytes/macrophages weredisproportionately depleted in spinal cords of CXCL13−/− mice duringlate stages of disease, such that the absolute number ofCD45hiCD11b^(hi) cells was, on average, 18 fold lower in CXCL13−/− thanin WT cords (FIG. 14). A similar trend was observed with respect toCD11c+ cells.

162. Interestingly, CXCR5 was expressed on similar percentages ofCNS-infiltrating cells in CXCL13−/− and WT mice. At peak disease, 10-20%of both CD4+ and CD4− T-cells were CXCR5+ irrespective of host genotype.Furthermore, the cellular composition of CXCR5+ cells did not differ. Inboth groups, approximately 50% of CXCR5+ cells were CD3+ T-cells, andthe other 50% included B cells, NK cells, and a subset of myeloid cells.

Discussion

163. Herein it was demonstrated for the first time that the lymphoidchemokine CXCL13 plays a pathogenic role in EAE, a Th1-mediated, organspecific autoimmune disease. CXCL13 first appears in spinal cords ofaffected mice during the presenting episode and its levels rise steadilyas the disease progresses (FIGS. 1, 2). The experiments with CXCL13deficient mice, as well as with wildtype mice treated with anti-CXCL13neutralizing antibodies, demonstrate that the chemokine contributes todisease severity within days of symptom onset. However, it also exertssignificant effects during later stages, as reflected by the decreasedrate of relapsing and chronic EAE in CXCL13−/− animals (FIG. 5 a, Table1). In corroboration with these clinical results, spinal cords fromCXCL13−/− mice have relatively mild inflammatory infiltrates and containa decreased percentage of myeloid cells by comparison to their wildtypecounterparts. Furthermore, pathological changes in CXCL13 deficient miceare confined, in large part, to the subpial white matter, whereasmultiple deep white matter tracts are affected in wildtype mice.

164. The finding that the lymphoid chemokines, CXCL13, CCL21 and CCL19are upregulated in the inflamed CNS of mice with EAE is supported byseveral recent publications from other laboratories. Magliozzi et al.reported that CXCL13 transcripts are elevated in the CNS ofPLP₁₃₅₋₁₅₁-immunized SJL mice during exacerbations, although not duringremissions (Magliozzi, R., et al. 2004. J Neuroimmunol 148:11-23). Bycontrast, it was found that CXCL13, CCL21 and CCL19 levels rise steadilyduring disease progression, including the remission phase between thefirst and second exacerbations, as measured by quantitative RPA. Similarresults were obtained in independent experiments using semi-quantitativeRT-PCR. The discrepancy between the findings herein and those ofMagliozzi and colleagues might be the result of methodological orsampling differences. Nonetheless, in all studies published thus far,CNS lymphoid chemokine levels were found to reach their height duringrelapsing or progressive stages of EAE, suggesting an associationbetween disease chronicity and chemokine expression within the targetorgan (Alt, C., et al. 2002. Euro J Immunol 32:2133-2144,Columba-Cabezas, S., et al. 2003. Brain Pathology 13:38-51, (Magliozzi,R., et al. 2004. J Neuroimmunol 148:11-23).

165. CXCL13 is produced by mesenchymal stromal cells in secondarylymphoid tissues (Cyster, J. G. 1999. Science 286:2098-2102). However,lymphoid chemokines can also be secreted by myeloid cells (Ishikawa, S.,et al. 2001. J Exp Med 193:1393-1402, Ansel, K. M., et al. 2002.Immunity 16:67-76). The data pinpoint CD11b+CD11c+ myeloid cells as thecellular source of CXCL13, as well as CCL19 and CCL21, in the inflamedCNS. Consistent with these results, Columba-Cabezas and colleaguesdetected CCL19 in infiltrating leukocytes, which they suspected weremacrophages and dendritic cells, in EAE lesions (Columba-Cabezas, S., etal. 2003. Brain Pathology 13:38-51). The findings do not exclude thepossibility that non-hematopoetic cells also contribute to lymphoidchemokine production in the CNS during the course EAE. Based on theRT-PCR study shown in FIG. 4 a it appears that CCL19 and CCL21 mRNAexpression declines in spinal cord mononuclear cells between the firstand second episode of relapsing disease, whereas levels of bothchemokines rise in whole spinal cord tissues harvested at the same timepoints.

166. CXCL13 has a significant impact on the severity of inflammatory CNSdemyelination and its clinical manifestations. Although lymphoidorganogenesis is impaired in CXCL13−/− mice, their resistance to EAEcannot be attributed to a failure to activate autoreactive T-cells inthe periphery. In fact, splenocytes from MOG-immunized CXCL13−/− micemount significant antigen-specific IL-2 and IFNγ responses upon ex vivochallenge, indicating that autoreactive T-cell priming, clonal expansionand differentiation does occur in the absence of CXCL13. Furthermore,CXCL13 deficient mice develop less severe EAE than wildtype counterpartsfollowing the transfer of myelin-reactive T-cells that had been primedin chemokine-sufficient donors (FIG. 5 b). In parallel experiments itwas shown that αCXCL13 neutralizing antibodies suppress adoptivelytransferred EAE in highly susceptible SJL mice (FIG. 5 c). Collectively,the data indicate that CXCL13 plays a unique role during the effectorphase of the disease, by altering the biological activities of CNSinfiltrating CXCR5+ cells.

167. The vast majority of CXCR5+cells in spinal cord infiltrates of micewith acute EAE are CD4+CD3+ T-cells. CXCR5+ B cells are present as well,but in sparse numbers. Although CXCL13 plays an indispensable role inthe formation of organized B cell follicles in lymph nodes and spleen,the chemokine is not necessary for B or T-cell accumulation in theseorgans per se (Forster, R., et al. 1996. Cell 87:1037-1047, Ansel, K.M., et al. 1999. J Exp Med. 190:1123-1134). It was shown that CXCL13alters the course of EAE by modulating the biological activities ofCXCR5+ cells once they have infiltrated the CNS, rather than byattracting them across the blood-brain-barrier initially. Similarconclusions were reached regarding the effects of CXCL13 on CD4+CD3−lymphoid inducer cells during lymphoid organogenesis. While CD4+ CD3−cells fail to induce Peyer's patches in CXCR5 deficient mice, theyaccumulate in large numbers in the mesenteric lymph nodes and spleen.However, in the absence of CXCL13 simulation the inducer cells fail toexpress an activated form of α4β1 integrin, which is necessary forinteractions with stromal cells that lead to the induction of intestinallymphoid tissues (Finke, D., et al. 2002. Immunity 17:363-373).

168. By analogy to its role in the organization of B cell follicles, itis shown herein that CXCL13 coordinates the positioning ofmyelin-specific T-cells in relationship to other leukocyte subsetswithin perivascular infiltrates in a manner that facilitates cognatecell-to-cell interactions. The data indicates that CXCL13 is produced byCD11b+CD11c+ myeloid cells within the inflamed spinal cord (FIG. 4).Such cells could serve as APCs for reactivation of myelin-reactiveT-cells following their passage across the blood-brain barrier. If so, aCXCL13 gradient could draw newly arrived CXCR5+ T-cells directly to APCsbearing myelin peptide/MHC Class II complexes, thereby facilitatingT-cell reactivation in the target organ. A recent study underscored theimportance of T-cell reactivation within the CNS for the production ofmonocyte chemoattractants, recruitment of macrophages and, ultimately,the clinical manifestation of EAE (Aloisi, F., et al. 2000. J. Immunol.164:1705). Interestingly, the histopathological and FACS studiesdemonstrate impaired accumulation of macrophages in EAE lesions inCXCL13−/− mice, possibly reflecting insufficient reactivation ofmyelin-reactive T-cells.

169. CXCL13 might also facilitate collaborations between T and Blymphocytes within the CNS. Cognate T-B cell interactions are known tooccur in the CNS of patients with MS, leading to antigen-driven B cellclonal expansion and antibody production in situ (Baranzini, S. E., etal. 1999. J Immunol 163:5133-5144, Correale, J., and de los MilagrosBassani Molinas, M. 2002. J of Neurology 249:375-389, Colombo, M., etal. 2000. J Immunol 164:2782-2789, Gerritse, K., et al. 1994. JNeuroimmunol 49:153-159). Magliozzi and colleagues identifiedlymphoid-follicle like structures containing B cells within the meningesof mice undergoing progressive and chronic-relapsing EAE (Magliozzi, R.,et al. 2004. J Neuroimmunol 148:11-23). As has been proposed in otherautoimmune models, CXCL13 might bring autoreactive T helper and B cellsinto close proximity to one another. Ultimately, this could result inthe local production of pathogenic antibodies, which could facilitatedemyelination (Cross, A. H., et al. 2001. J Neuroimmunol 112:1-14,Lyons, J. A., et al. 1999. European J Immunol 29:3432-3439, Genain, C.P., et al. 1995. Journal of Clinical Investigation 96:2966-2974).

170. In later phases of EAE, the pathogenic effects of CXCL13 might berealized through alternative routes. Previous publications haveemphasized the potential role of CXCL13 in lymphoid neogenesis in animalmodels. Transgenic expression of CXCL13 under the rat insulin promoterresults in the formation of lymph node-like structures in the pancreascharacterized by lymphoid-myeloid aggregates, MAdCAM-1+ blood vesselsand local induction of CCL21 (Luther, S. A., et al. 2000. Immunity12:471-481). These same features have been observed in EAE and/or MSlesions and most likely participate in the perpetuation of chronic orrelapsing CNS inflammation (Cross, A. H., et al. 1990. Lab Invest63:162-170, Raine, C. S., et al. 1980. Laboratory Investigation43:150-157, Raine, C. S., et al. 1984. Laboratory Investigation51:534-546, Prineas, J. W., and Wright, R. G. 1978. LaboratoryInvestigation 38:409-421, Prineas, J. W. 1979. Science 203:1123-1125,Kanwar, J. R., et al. 2000. J Neuroimmunol 103:146-152, Alt, C., et al.2002. Euro J Immunol 32:2133-2144, Columba-Cabezas, S., et al. 2003.Brain Pathology 13:38-51). Furthermore, CXCL13 is present in thesynovial tissues of patients with rheumatoid arthritis and salivaryglands of patients with Sjogren's syndrome in association with organizedB-T-cell aggregates that resemble lymphoid follicles (Shi, K., et al.2001. J Immunol 166:650-655, Salomonsson, S., et al. 2002. Scan JImmunol 55:336-342). With the current study, it was demonstrated thatCXCL13 was induced in CNS tissues following infiltration bymyelin-reactive T-cells.

171. During the formation of secondary lymphoid organs, CXCL13stimulates CD4+CD3− lymphoid tissue inducer cells to express anactivated form of α4β1 and membrane lymphotoxin-α1β2 (Finke, D., et al.2002. Immunity 17:363-373) both of which are necessary for criticalinteractions with stromal cells. CXCR5+IL-7Rα+CD4+ CD3− cells weredetected, indicative of lymphoid inducer cells, in EAE infiltrates.

172. The current study is the first to directly demonstrate anon-redundant role of CXCL13 in an organ-specific autoimmune disease.Furthermore, it introduces the concept that CXCL13 can participate inthe pathogenesis of autoimmune conditions that are traditionallyconsidered to be CD4+ Th1 cell driven, as well as in those primarilymediated by autoantibodies. The data indicate that agents thatneutralize CXCL13 or block its receptor can be useful in the treatmentof human autoimmune conditions, such as multiple sclerosis.

Example 2 Expression of Lymphoid Chemokine/Chemokine Receptor mRNA inSpinal Cords of Mice with EAE

173. To determine whether the lymphoid chemokines, CXCL13, CCL19, andCCL21 are expressed in the CNS during EAE, RT-PCR was performed on RNAextracted from spinal cords from symptomatic mice and naïve controls.B10.PL mice (n=5) were actively immunized with an immunodominant peptideof myelin basic protein (MBP_(Ac1-11)) in Complete Freund's Adjuvant(CFA). At the time of sacrifice, three mice were in relapse (lanes 1, 3,4), one mouse was experiencing the first clinical episode of disease(lane 2), and one mouse remained asymptomatic (lane 5). CXCL13 mRNA wasexpressed in spinal cords from the four mice with EAE, but not inasymptomatic or naïve animals (FIG. 1A). CXCR5 mRNA (the receptor forCXCL13) was detectable in two of the diseased cords. CCL19 mRNA was alsopresent in the diseased spinal cords and, at lower levels, in spinalcords from naïve animals. The expression of CCR7 (the receptor for CCL19and CCL21) roughly followed the same pattern. mRNA for CXCL13, CCL19,CCL21, CXCR5 and CCR7 was detected in spinal cords from SJL mice andC57BL/6 mice with EAE induced using peptides of proteolipid protein(PLP) and myelin oligodenrocyte glycoprotein (MOG), respectively. Hence,CNS upregulation of lymphoid chemokines appears to be a generalphenomenon in EAE, irrespective of strain or target autoantigen. CXCL13,CCL19 and CCL21 are not upregulated in spinal cords of mice injectedwith CFA alone.

174. Next levels of CXCL13, CCL21 and CCL19 mRNA in the CNS of miceduring the course of relapsing-remitting disease were measured using anRNase Protection Assay (RPA). SJL mice were actively immunized againstan immunodominant PLP epitope (PLP₁₃₉₋₁₅₁) in CFA and assessed daily forneurological deficits. Representative mice were sacrificed during thefirst episode, remission, and relapse of EAE, respectively, for mRNAquantification. It was found that expression of all three lymphoidchemokines rose progressively during the disease course (FIG. 2A).

175. FIGS. 1A and 2A demonstrate that lymphoid chemokine and chemokinereceptor mRNA are upregulated in the cords of mice with EAE induced byactive immunization with myelin peptides in combination with adjuvants(CFA and Bordetella pertussis toxin). In previous studies it was foundthat draining lymph node (LN) cells from SJL mice immunized with PLPpeptide in IFA (without heat killed Mycobacteria) only induce EAE innaïve recipients following antigenic challenge in the presence ofrecombinant IL-12 (Segal, B. M., et al. 2000. J Immunol 164:5683-5688,Bagaeva, L. V., et al. 2003. J Neuroimmunol 137:109-116). FIG. 1Bdemonstrates that spinal cords harvested from mice with passivelytransferred EAE (12 days following injection of PLP/IFA primed, IL-12stimulated T-cells), but not healthy controls, express CXCL13 and CXCR5.Furthermore CCL19 and CCR7 mRNA are also upregulated in the CNS ofsymptomatic recipients of encephalitogenic T-cells by comparison tocontrol mice.

176. FIGS. 1 and 2 demonstrate that lymphoid chemokines are present inspinal cords of mice with EAE on the mRNA level. In order to testwhether this mRNA is translated into protein, Western blot analyses wasperformed on pooled spinal cords from 10 mice with adoptivelytransferred EAE (mean clinical score 2.5) and 10 healthy controls.Spinal cord lysates from each group were immunoprecipitated usingheparin Sepharose, as previously described (Luther, S. A., et al. 2000.PNAS 97:12694, Ansel, K. M., et al. 2000. Nature 406:309-314, Luther, S.A., et al. 2002. J Immunol 169:424-433). The chemokine proteins weredetected with goat anti-mouse CCL19, CCL21 or CXCL13 (R&D Systems,Minneapolis, Minn.) followed by anti-goat horseradish peroxidase (PierceEndogen); development was with Supersignal West Femto MaximumSensitivity Substrate (Pierce). Recombinant chemokines and spleenlysates were used for positive controls. CXCL13, CCL21 and CCL19proteins were readily detected in cords from the mice with EAE, but nothealthy controls (FIG. 1C). Lysates from symptomatic and healthy miceyielded similar amounts of β-actin, a housekeeping protein. Similarresults were obtained with cords from MOG-sensitized C57BL/6 mice.

177. In several EAE models intrathecal CXCL13 and CCL19 were found to beassociated with MHC class II transactivator (CIITA) form I, a dendriticcell-specific molecular marker (FIG. 2A, Suter, T., et al. 2000. Eur. J.Immunol. 30:794). This indicates that dendritic cells are present inrelapsing EAE lesions. Dendritic cells could be attracted toinflammatory foci by lymphoid chemokines (Cyster, J. G. 1999. Science286:2098-2102, Moser, B., and Loetscher, P. 2001. Nature Immunology2:123-128, Campbell, J. J., et al. 1998. J. Cell. Biolo. 141:1053). Onthe other hand, myeloid dendritic cells have been shown to produce CCL19as well as CXCL13 under some circumstances (Vissers, J. L. M., et al.2001. Eur. J. Immunol. 31:1544, 26). GM-CSF drives the differentiationof CNS microglia into myeloid DC in vitro (Fischer, H. G. and Reichmann,G. 2001. J Immunol. 166:2717, Santambrogio, L., et al. 2001. PNAS98:6295, Aloisi, F., et al. 2000. J. Immunol. 164:1705). Interestingly,GM-CSF is commonly produced by activated T-cells includingmyelin-reactive CD4+ T-cell lines (Wong, R. L., et al. 1989. Cell.Immunol. 123:445). Furthermore, it is expressed in the inflamed cords ofmice with EAE in association with CD3 (FIG. 1A). Hence, GM-CSF, secretedby myelin-reactive T-cells in EAE lesions, can stimulate microglia todifferentiate into myeloid dendritic cells and produce lymphoidchemokines locally.

178. In order to assess whether dendritic-like cells accumulate in EAElesions in association with infiltrating T-cells, frozen or whole mountsections of spinal cords from symptomatic mice were stained with amonoclonal antibody against CD11c, a myeloid dendritic cell marker.CD11c⁺ cells were readily detected within perivascular infiltrates.Furthermore, these dendritic-like cells formed clusters with CD4⁺T-cells (FIG. 8).

179. In an initial attempt to identify the cellular source of lymphoidchemokines in the CNS during EAE, mononuclear cells from spinal cords ofsymptomatic mice were isolated over a 30%/70% Percoll gradient. Flowcytometric analysis revealed that these cells were bone marrow derived(95% of the total cell population was CD45⁺) and consisted primarily ofmyeloid cells, many of which express the dendritic cell marker CD11c(FIG. 4A). Lymphoid cells were almost exclusively CD4⁺ T-cells, andcomprised 26% of the total population. RT-PCR studies revealed thatCD11c⁺CD11b⁺ spinal cord inflammatory cells, purified from mice at peakdisease, express CXCL13, CCL21 and CCL19 mRNA (FIG. 4B). Interestingly,while CXCL13 mRNA levels appear to rise in the CD11c⁺ cells between thefirst and second EAE episode, CCL19 and CCL21 mRNA levels wane.

180. The results shown in FIGS. 1A, and 2A indicate that CXCL13 isproduced in the CNS during clinical EAE and that its receptor, CXCR5, isexpressed on the mRNA level concurrently. CXCR5 is expressed on a subsetof CD4⁺ T-cells in germinal centers in peripheral lymphoid tissues (Kim,C. H., et al. 2001. J Exp Med. 193:1373-1381). Collectively, theseobservations led to the belief that myelin-reactive effector T-cellsmight express CXCR5, thereby facilitating their migration fromperipheral sites to active demyelinating lesions, altering theirlocalization within inflammatory foci in the central nervous system(possibly to faciliate interactions with B cells or myeloid cells),and/or facilitating their stimulation within the central nervous system.If so, PLP-primed LN cells used to induce EAE in adoptive transferstudies might include CXCR5⁺ CD3⁺ T-cells. To investigate thatpossibility, PLP-primed LN cells were harvested 4 days after antigenicstimulation in vitro and permeabilized them prior to staining withFITC-labeled anti-CXCR5 monoclonal antibodies and analysis by flowcytometry. A control sample was labeled with isotype matched,FITC-labeled, antibodies. As shown in FIG. 9, a subset of CD3+ T-cellsamong the PLP-primed LN cells was indeed positive for CXCR5. Similarresults were obtained by staining for CXCR5 on the cell surface.

181. In order to measure CXCR5+T-cell accumulation in the CNS during EAEspinal cord mononuclear cells were isolated from mice during the firstexacerbation and stained them with flourochrome-labeled monoclonalantibodies specific for CXCR5 and either CD4 or CD3. Subsequent flowcytometric analysis revealed that between 11-16% of CNS inflammatorycells were CD4⁺ CXCR5⁺. A representative example is shown in FIG. 5C.Virtually all of these cells bore the memory cell marker CD44 andexpressed iCOS. (This cell surface phenotype is typical of germinalcenter follicular helper CD4+ T-cells that promote B celldifferentiation. The percentage of CD4⁺CXCR5⁺ T-cells within the CNSmononuclear cell compartment increases as the disease advances. By thesecond relapse they comprise 23-25% of spinal cord mononuclear cells.Although IgM+ CXCR5+ B cells only comprise a minor subset of CNSinflammatory cells during the first exacerbation, they accumulate ingreater numbers in later stages.

182. The data shown in FIG. 3 demonstrates that CXCR5⁺ CD4⁺ T-cellsinfiltrate the CNS in correlation with local CXCL13 expression. Theimpact of CXCL13 neutralization on clinical EAE was assessed. SJL micewere treated with goat anti-mouse CXCL13, isotype matched controlantibodies or PBS (n=5/group) on days 3, 6 and 10 post transfer. Animalswere examined daily and rated for neurological impairment on a 5 pointscale. As shown in FIG. 5, the animals treated with anti-CXCL13 wereprotected from EAE.

183. In a parallel approach to that illustrated in FIG. 5, C57BL/6wildtype and CXCL13 deficient mice were immunized with MOG₃₅₋₅₅ in CFAand rated them daily for neurological deficits. Although CXCL13−/− micesuccumbed to clinical EAE, the intensity of their symptoms wassignificantly reduced and they recovered at a faster rate and morecompletely than their wildtype counterparts.

184. CXCL13 deficient mice have disorganized splenic architecture aswell as a paucity of certain lymph nodes. Therefore, resistance of thesemice to EAE can results from defective priming of autoimmune effectorcells in the periphery as opposed to defective recruitment and/orretention of CXCR5⁺ T-cells in the CNS. To address that possibilityMOG-specific responses of splenocytes from mice were measured by anumber of T-cell assays. It was found that sensitized CXCL13 deficientanimals mounted significant lymphoproliferative, IFNγ and IL-2 responseson ex vivo challenge that were comparable to those of their wildtypecounterparts (Table 1). In fact, MOG-specific proliferation appeared tobe enhanced in CXCL13−/− mice.

Example 3 CXCR5⁺ and CCR7⁺ Leukocytes Accumulate in EAE Lesions

185. It has been shown that CCR7⁺ and CXCR5⁺ leukocytes accumulate inthe CNS during the course of EAE. Alt and colleagues detected CCR7⁺cells in perivascular EAE infiltrates by in situ hybridization andimmunoshitochemical analysis. Furthermore, it was found that CCR7⁺ cellsappear in spinal cords of sick mice by RT-PCR (FIG. 1A). In addition,CXCR5⁺ CD4⁺ T-cells accumulate in the CNS as early as the first episodeof EAE (FIGS. 1A, 2A, and 3). The infiltrating T-cell population becomesincreasingly enriched for CXCR5⁺ and CCR7⁺ cells during diseaseprogression, in conjunction with rising levels of CXCL13 and CCL19 (FIG.2A). CXCR5⁺ IgM⁺ B cells begin to appear in the CNS during the firstrelapse in SJL mice and during the chronic phase of EAE in C57BL/6 mice.A population of CD4, CD3, CCR7⁺ cells that can represent mature CD11c⁺dendritic cells arise at an early time point in the clinical course.

186. The chemotaxis of spinal cord mononuclear cells harvested from micewith EAE across a transwell membrane in response to a CXCL13, CCL19 orCCL21 gradient (Legler, D. F., et al. 1998. J. Exp. Med. 187:665) can bemeasured. In addition, migrating cells can be rigorously characterizedby flow cytometric analysis. It has been found that CCR7⁺ and CXCR5⁺populations within CNS infiltrates evolve over the course of EAE. Forexample, CXCR5⁺ cells are almost exclusively CD4⁺ at the time of thefirst exacerbation in SJL mice, whereas CXCR5⁺ B cells begin toaccumulate by the subsequent relapse (FIG. 3). B cells also become moreprominent in chronic EAE infiltrates of MOG-sensitized C57BL/6 mice asthe disease advances.

187. The lower chamber of a transwell (Costar; 5.0 μm pore sizefilters/6.5 mm diameter) can be filled with tissue culture media with orwithout recombinant CXCL13, CCL19 or CCL21 (R&D Systems) over a range ofconcentrations (between 5 and 100 nM in a total volume of 0.6 ml). Theseconcentrations were chosen based on previous reports of optimalconditions for stimulating chemotaxis (Legler, D. F., et al. 1998. J.Exp. Med. 187:665). CNS mononuclear cells (enriched over a Percollgradient) can be added to the upper chamber (1.5×10⁶ cells in 0.25 ml).After a 3 hour incubation at 37° C., 5% CO₂, cells can be harvested fromthe lower chamber, concentrated by centrifugation, counted and stainedwith FITC- and/ or PE-labeled antibodies specific for selected leukocytemarkers (such as CD4, CD8, IgM, B220 and CD11c). In certain instancescells can be costained to measure memory markers (CD62L, CD44),costimulatory molecules (iCOS, CD40, CD40L), or cytokine receptors(IL-7R) The samples can then be subjected to flow cytometric analysis.Whole splenic cell suspensions or purified splenic subpopulations canserve as positive controls (B cells for CXCL13; T-cells forCCL19/CCL21). Each assay can be done in triplicate. In certainexperiments neutralizing antibodies can be added along with recombinantchemokines to some wells to further demonstrate the specificity ofchemotaxis.

188. Data can be presented as the number of migrated cells (either totalor of a particular subset) into chemokine-containing wells minus thenumber of migrated cells into wells containing media only. Means and pvalues can be determined by ANOVA with parametric, 2-tailed post-hocanalysis. This approach can allow in detail characterization of the cellsurface phenotype of CNS mononuclear cells that are responsive toindividual lymphoid chemokines during different stages of EAE.

189. CXCL13 can stimulate CXCR5⁺CD4⁺ T-cells in inflammatorydemyelinating lesions. Furthermore, these cells can co-express iCOS,IL-7 receptor and CD40L, a profile reminiscent of germinal centerfollicular helper T-cells. At later stages of disease, IgM⁺ CXCR5⁺ Bcells can comprise a larger percentage of CXCL13-responsive cells withinthe CNS infltrates. On the other hand, CCL19 and CCL21 can stimulateCCR7⁺CD4⁺ T-cells with a resting or Tcm phenotype as well as CCR7⁺CD11c⁺ myeloid cells.

190. During later stages of EAE in C57BL/6 mice a growing population ofCXCR5⁺CD3⁻ cells has been detected, at least some of which appear to beB cells. This subpopulation can be characterized in detail by costainingfor B220, immunoglobulin light chains, CD19, CD4 and CD40. The relativedependence of CXCR5⁺ subsets on CXCL13 for CNS accumulation can bedetermined by performing flow cytometric analysis on spinal cordmononuclear cells from wildtype and CXCL13 deficient mice following thetransfer of encephalitogenic T-cells.

191. Analogous experiments can be performed using plt/plt mice to assessthe role of CCL21 and CCL19 on the recruitment of CCR7⁺ leukocytes. Onceagain an adoptive transfer model can be used. For flow cytometricanalysis, infiltrating CCR7⁺ CD4⁺ T-cells can be costained withantibodies against CD62 and CD44 in order to distinguish naïve andcentral memory lymphocytes. Mononuclear cells against CD11c and CD11bcan be stained in an attempt to detect mature CCR7⁺ dendritic cells thatare responsive to CCL19/21.

192. These studies can indicate that CCL19/21 can be required foroptimal accumulation of CD11c⁺ myeloid cells and central memory andnaïve T-cells in CNS infiltrates. On the other hand, CXCL13 promotes therecruitment of activated CD4⁺ T-cells (with a cell surface phenotypecharacteristic of follicular helper cells) and B cells.

193. The data disclosed herein indicate that, although lymphoidchemokine production begins in the CNS during the first exacerbation,levels rise substantially at relapse (FIG. 2A). Therefore, CD4⁺ T or Bcells expressing a genetic marker can be transferred into activelyimmunized mice during their first remission of EAE. The homing patternsof the transferred cells can then be followed during the subsequentrelapse in hosts injected with either neutralizing antibodies againstlymphoid chemokines or control antibodies. It is understood thatlymphoid chemokine neutralizing antibodies can block the homing oftransferred cells to the CNS.

194. EAE infiltrates contain a substantial number of T-cells thatexpress a naïve phenotype (Raine, C. S., et al. 1984. LaboratoryInvestigation 51:534-546). Many naïve T-cells express CCR7, the receptorfor CCL19 and CCL21 (Cyster, J. G. 1999. Science 286:2098-2102, Luther,S. A., et al. 2002. J Immunol 169:424-433). Therefore, the effects ofCCL19 and CCL21 blockade on the migration of resting T-cells to the CNSduring EAE relapses in SJL mice can be assessed. For example, Thy1.2⁺SJL mice can be used as donors and congeneic SJL Thy1.1⁺ mice asrecipients. The latter mice are obtained from Jackson Laboratories. Theyhave been tested extensively to show a lack of alloreactivity to theThy-1 disparity. Following injection donor cells can be identified bystaining with antibodies specific for the Thy 1.2 marker.

195. CD4⁺ T-cells expressing a naïve phenotype (CD62^(high), CD44^(low))can be purified from pooled lymph nodes and spleens of unmanipulated Thy1.2⁺ donors by FACS sorting. These cells will then be injected intoThy1.1⁺ mice during their first remission of EAE (defined as a clinicalscore of 1 or 2 points below peak severity maintained for 2 or moredays). Hosts can be divided into two groups, one of which can be treatedwith a cocktail of neutralizing antibodies against CCL19 and CCL21 (R&DSystems) and the other with isotype matched control antibodies (250 μgof each mAb i.p./mouse every 72 hours from the time of cell transferonwards). This dosing protocol has been previously used to neutralizeCCL19 and CCL21 in vivo in other experimental systems with significantbiological effects (Itakura, M., et al. 2001. J Immunol. 166:2071). Micecan then be sacrificed at the time of expected peak relapse. Mononuclearcells can be isolated from their spinal cords and stained against CD4and Thy1.2 prior to flow cytometric analysis.

196. As mentioned above, a subset of activated CD4⁺ T-cells and B cellshome to the CNS during relapsing EAE in response to CXCL13. To testthis, PLP-reactive CD4⁺ T-cell lines derived from Thy1.2⁺ SJL donors canbe injected into Thy1.1⁺ hosts during the first remission of activelyinduced EAE. In other experiments Thy1.2⁺ IgM⁺ CD19⁺ B cells, isolatedfrom naïve splenocytes using magnetic beads or FACs sorting, can betransferred. Subsequently, the hosts can be treated with either aneutralizing antibody against CXCL13 or isotype matched controlantibodies (R&D Systems, 200 μg/mouse i.p. every 72 hours, FIG. 5;(Itakura, M., et al. 2001. J Immunol. 166:2071) until sacrifice at thetime of expected relapse. CNS-infiltrating mononuclear cells can then beisolated and analyzed for the presence of Thy1.2⁺ CD4⁺ or Thy1.2⁺ CD19⁺emigrants. It is understood from the experiments above thatanti-CCL19/21 antibodies can block the migration of naïve cells(including those reactive to secondary myelin epitopes) and anti-CXCL13antibodies can block the migration of primed myelin-reactive T-cells andB cells to the CNS during EAE relapses.

Example 4 Lymphoid Chemokines Play a Physiological Role in theEstablishment of Progressive and Relapsing EAE

197. Chemokine deficient mice can be used to assess the contribution ofCXCL13, CCL19 and CCL21 to disease severity and chronicity. CXCL13deficient mice on a C57BL/6 background (Ansel, K. M., et al. 2000.Nature 406:309-314, Ansel, K. M., et al. 2002. Immunity 16:67-76) andplt mice (which are deficient in CCL19 and CCL21 in secondary lymphoidtissues), also on a C57BL/6 background, were obtained (Luther, S. A., etal. 2000. PNAS 97:12694, Mori, S., et al. 2001. J. Exp. Med. 193: 207,Gunn, M. D., et al. 1999. J. Exp. Med. 189:451-460, Nakano, H., andGunn, M. D. 2001. J Immunol. 166:361).

198. In the experiments disclosed herein it was found that clinical EAEwas suppressed in actively immunized CXCL13−/− mice. However, secondarylymphoid tissues were disorganized in these knock-outs as well as in pltmice. Plt mice demonstrate disrupted homing of naïve T-cells andactivated dendritic cells to T-cell zones and an abnormal distributionof T-cells within secondary lymphoid tissues (Gunn, M. D., et al. 1999.J. Exp. Med. 189:451-460). CXCL13 deficient mice have absent or verysparse peripheral lymph node tissues, a reduced number of Peyer'spatches and they lack follicular dendritic cells and organized follicles(Gunn, M. D., et al. 1998. Nature. 391:799, Ansel, K. M., et al. 2000.Nature 406:309-314). Such abnormalities of secondary lymphoid tissuemight interfere with the priming and/or expansion of antigen-specificT-cells in vivo in response to vaccination. An adoptive transfer modelin which wildtype myelin-reactive T-cells are injected into naïve,syngeneic chemokine-deficient or wildtype recipients allows for theavoidance of artifacts arising from insufficient effector cellactivation, differentiation and/or expansion secondary to lymphoidchemokine deficiencies during T-cell priming.

199. C57BL/6 mice are relatively resistant to EAE induced by theadoptive transfer of myelin-primed T-cells according to standardprotocols. However, it has been shown that myelin oligodendrocyteglycoprotein (MOG)-specific T-cells from primed C57BL/6 donors acquireencephalitogenic properties following antigenic challenge in thepresence of recombinant IL-12 (Spahn, T. W., et al. Eur. J. Immunol. 29:4060). Therefore, this approach can be used to compare the effectorphase of EAE in chemokine-deficient and wildtype hosts. In eachexperiment donor cells can be cultured with an optimal concentration ofMOG peptide (25 μg/ml) and murine IL-12 (5 ng/ml) in TCM. At 96 h, thecells can be harvested, washed, counted and injected into sex and agematched wildtype and chemokine deficient recipients (50×10⁶ cells/mousei.p.). The disease incidence, chronicity, relapse rate, severity andhistological features can be compared between the experimental groups.The significance of observed differences can be assessed using eitherStudent's t test or the Wilcoxon signed rank test. It is understood thatCXCL13−/− and plt mice can experience a relatively mild from of EAE withmore complete recovery from exacerbations.

200. One of the ways in which CNS lymphoid chemokines can facilitateclinical EAE is by organizing white matter infiltrates through theprocess of lymphoid neogenesis. In fact, mice that express a CXCL13 orCCL21 transgene under control of the rat insulin promoter developlymph-node like structures in the pancreas that contain T and B cellzones, high endothelial venules and stromal cells. Transgenic CXCL13also induced CCL21 expression in inflamed blood vessels and stromalcells within these lymphoid structures.

201. A similar phenomenon can occur during the development of whitematter infiltrates during EAE, driven by the production of endogenouslymphoid chemokines in the CNS. Indeed, it has been found that MAdCAM-1is upregulated on cerebrovascular blood vessels in inflamed cords ofwildtype mice with EAE (FIG. 10). Furthermore, a subpopulation ofCXCR5⁺α4β7⁺ IL-7R⁺CD4⁺CD3⁻ cells were detected among spinal cordmononuclear cells that could represent lymphoid precursor cells(mesenchymal cells that fix sites of lymphoid organogenesis). Hence,spinal cords from wildtype and lymphoid chemokine deficient adoptivetransfer recipients can be compared for features of lymphoid neogenesisand for accumulation of lymphoid precursor cells.

202. Spinal cords can be removed from wildtype, CXCL13 deficient, andplt/plt mice with adoptively transferred EAE across a range of clinicalscores. An attempt can be made to match animals across groups based upondegree of paralysis. Representative cords can be snap frozen, sectionedand stained for BP3 and ER-TR7 (Accurate Chemicals), antigens common tostromal cells in B and T-cell areas of lymphoid organs. This approachwas used to detect a network of stromal cells in the pancreaticinfiltrates of RIP-CXCL13 transgenic mice. Contiguous sections can bestained with antibodies specific for HEV-like adhesion moleculesincluding MAdCAM-1 and the peripheral lymph node addressin, PNAd.Immunoreactive cells can be counted in spinal cord sections from 6-8different areas by two blinded examiners at ×10 and ×63 magnification.Lastly, CCL21 expression in cords from CXCL13 deficient can be comparedto wildtype mice by RPA and immunohistochemistry.

203. Accumulation/modulation of lymphoid organ precursor cells to theCNS: Lymphoid organ precursor cells (bearing the cell surface phenotypeCD4⁺CD3⁻IL-7Rα⁺) fix sites of future lymph node organogenesis duringearly development. These cells are CXCR5⁺CCR7⁺ and are drawn toperipheral lymphoid areas by CXCL13 and/or CCL19/CCL21. Although CXCL13and CCL19/21 have overlapping functions in attracting the precursorcells they are not completely redundant. That lymphoid precursor cellsare recruited into the CNS during EAE by lymphoid chemokines can betested using the following strategies: (i) Flow cytometric studies:Spinal cord mononuclear cells from wildtype, CXCL13−/− and plt/pltadoptive transfer recipients can be pooled and costained with antibodiesagainst CD4, CD3, IL-7 receptor, α4β7 integrin and/or CXCR5 or CCR7prior to analysis on the flow cytometer. Cells expressing a lymphoidprecursor cell surface profile can be profiled in each group; (ii)RT-PCR for Id2 and RORγ transcription factors that are expressed bylymphoid precursor cells: Transcripts for Id2, a helix-loop-inhibitor,and RORγ a retinoic acid receptor-related orphan receptor, have beendetected in CD4⁺CD3⁻IL-7Rα⁺ lymphoid precursor cells. Furthermore, bothof these transcription factors appear to be critical for the generationand/or survival of the precursor cells. (CD4⁺CD3⁻IL-7Rα⁺CD45⁺ cells areabsent in embryonic intestines of mice that are genetically deficient ineither Id2 or RORγ. Both knock-outs lack lymph nodes and Peyer'spatches.) Consequently, Id2 or RORγ can be used as markers of lymphoidprecursor accumulation in the CNS. Spinal cords can be harvested fromwildtype and lymphoid chemokine deficient mice at serial time pointsfollowing adoptive transfer of encephalitogenic T-cells. RNA can beextracted from individual cords and perform RT-PCR with primers specificfor Id2, RORγ and β-actin. CXCL13 and CCL19/21 deficient mice may notsupport lymphoid neogenesis in the CNS during EAE and CD4⁺CD3⁻IL-7Rα⁺cells can fail to accumulate in white matter lesions.

204. There is an extensive literature suggesting that antibodies areproduced within the CNS of patient with MS. Furthermore, analysis of VDJtranscripts from cerebrospinal fluid cells or brain autopsy specimenssuggests that B cells undergo somatic hypermutation and terminaldifferentiation within the CNS. The data disclosed herein demonstratethat B cells also undergo isotype switching in the CNS during EAE. Inparticular, circle transcripts and mRNA encoding activation inducedcytidine deaminase (AID) has been detected in spinal cords from sickmice but not naïve controls (FIG. 11). Circle transcripts (CTγ), fromDNA loci excised during switch recombination, are a specific andimmediate byproduct of isotype switching in B cells and are considered ahallmark of active B cell activation. AID is a B cell-specific enzymethat is essential for somatic hypermutation and isotype switching. It isexpressed at high levels in germinal center B lymphocytes. Since CXCL13attracts CD40L⁺CD4⁺ T-cells and B cells to follicles and facilitatesgerminal center reactions, the chemokine can promote T-B cellcollaborations in white matter infiltrates during EAE.

205. CXCL13 deficient and syngenic wildtype mice can be sacrificed atserial time points following the transfer of encephalitogenic T-cells.Mice can undergo intracardiac perfusion with PBS prior to spinal cordharvest. mRNA can be extracted from individual spinal cords and performRT-PCR with primers for AID, Ig heavy chain circle transcripts(particularly CTγ2a, a circle transcript specific for the Th1 dependentIgG2a isotype), Mb-1 (a B cell-specific marker), CD4 and β-actin. RT-PCRcan be followed by Southern blot hybridization with internaloligonucleotide probes specific for the relevant molecules. Bandintensities can be measured by phosphorimaging and normalize AID andCTγ2a levels to Mb-1 (a measure of B cell infiltration). It isunderstood that AID and CTγ (circle transcript) expression can bereduced or absent in spinal cords from CXCL13 deficient mice.

Example 5 Monoclonal Antibody Against CXCL13 Ameliorates AdoptivelyTransferred EAE

2. Experiments with CXCL13 deficient mice and immunocompetent micetreated with polyclonal antibodies against CXCL13 indicated that thechemokine is important for the clinical manifestation of experimentalautoimmune encephalomyelitis (EAE) (FIG. 5 A-C). It was next questionedwhether anti-CXCL13 monoclonal antibodies would suppress EAE whenadministered during the effector phase of disease. To address thatquestion an adoptive transfer model was used in which naïve B10.PL micewere injected with activated, syngeneic T-cells that express atransgenic T-cell receptor specific for a peptide fragment of myelinbasic protein (MBP-TCR). MBP-TCR cells were stimulated with MBP peptideAc1-17 and recombinant murine IL-12 for 96 hours prior to transfer(35×10⁶ cells/recipient). The adoptive transfer recipients were thentreated with either a neutralizing monoclonal antibody against CXCL13 orisotype matched control antibody on days 3, 6 and 10 post transfer(n=5/group). As shown in FIG. 15, mice in the control group developed asevere form of EAE that peaked on day 11 with a mean clinical score of3.5, consistent with significant hindlimb paresis. By contrast, micetreated with the anti-CXCL13 monoclonal antibody experienced arelatively mild course with a mean score of 0.83 on day 11, indicativeof limp tail but no discernable limb weakness. These results indicatethat anti-CXCL13 monoclonal antibodies are effective therapies forinflammatory conditions including but not limited to multiple sclerosis.

206. Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains. Thereferences disclosed are also individually and specifically incorporatedby reference herein for the material contained in them that is discussedin the sentence in which the reference is relied upon.

207. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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1. A method of screening for an agent that inhibits a T-cell mediated inflammatory response in a subject with an inflammatory response comprising administering to the subject the agent and detecting the presence of CXCL13 in the subject, wherein a reduction in the level of CXCL13 as compared to a control level indicates an agent that inhibits the inflammatory response.
 2. The method of claim 1, wherein the agent is a neutralizing antibody to CXCL13.
 3. The method of claim 1, wherein the agent is an antibody to CXCR5 and wherein the antibody blocks CXCL13 binding without causing signaling through CXCR5.
 4. The method of claim 1, wherein the agent is a soluble form of CXCR5.
 5. The method of claim 1, wherein the inflammatory response comprises a response to an inflammatory condition.
 6. (canceled)
 7. The method of claim 1, wherein the inflammatory response comprises a response to a viral antigen.
 8. (canceled)
 9. The method of claim 1, wherein the inflammatory response comprises a response to a bacterial antigen.
 10. (canceled)
 11. The method of claim 1, wherein the inflammatory response comprises a response to a cancer antigen. 12-23. (canceled)
 24. A method of screening for an agent that inhibits a T-cell mediated inflammatory response in a subject, comprising the steps of a) administering the agent to the subject, b) inducing the inflammatory response in the subject, and c) detecting the presence of a chemokine selected from the group consisting of CXCL13, CXCR5, and CXCL13 and CXCR5 in the subject, wherein a reduction in the level of CXCL13, CXCR5, or both CXCL13 and CXCR5 in the subject as compared to a control level indicates an agent that inhibits an inflammatory response.
 25. The method of claim 24, wherein the agent is a neutralizing antibody to CXCL13.
 26. The method of claim 24, wherein the agent is an antibody to CXCR5 and wherein the antibody blocks CXCL13 binding without causing signaling through CXCR5.
 27. The method of claim 24, wherein the agent is a soluble form of CXCR5 or analog thereof. 28-47. (canceled)
 48. The method of claim 24, further comprising determining the antigen specificity of CXCR5 positive T-cells in the sample.
 49. The method of claim 24, further comprising determining the activation state of any CXCR5 positive T-cells in the sample.
 50. A method of treating a subject with an inflammatory condition comprising administering to said subject an effective amount of an agent that inhibits the interaction of CXCL13 and a receptor that binds to CXCL13, wherein the inhibition of said interaction of CXCL13 with said receptor reduces the inflammatory condition in said subject.
 51. The method of claim 50, wherein said inflammatory condition is a T-cell mediated autoimmune condition.
 52. The method of claim 50, wherein said agent comprises an antibody that specifically binds CXCL13.
 53. The method of claim 52, wherein said antibody prevents or inhibits the activity of CXCL13.
 54. The method of claim 52, wherein said antibody is a monoclonal antibody. 55-57. (canceled)
 58. The method of claim 50 wherein said agent is an antibody that specifically binds to CXCR5, and wherein said antibody blocks CXCL13 binding to CXCR5 without causing signaling through CXCR5.
 59. The method of claim 50, wherein said agent is a soluble form of CXCR5.
 60. (canceled)
 61. The method of claim 50, wherein said inflammatory condition is a neuroinflammatory condition. 62-64. (canceled)
 65. The method of claim 61, wherein said neuroinflammatory condition is selected from the group consisting of multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE), Guillain-Barre syndrome, Alzheimer's disease, transverse myelitis, acute disseminated encephalomyelitis, post-infectious encephalomyelitis, subacute sclerosing panencephalitis, and chronic inflammatory demyelinating polyradiculopathy.
 66. The method of claim 65, wherein said neuroinflammatory condition is multiple sclerosis. 67-68. (canceled)
 69. A method of reducing the exacerbation of an inflammatory condition in a subject comprising administering to said subject an effective amount of an agent that inhibits the interaction of CXCL13 and a receptor that binds to CXCL13, wherein the inhibition of said interaction of CXCL13 with said receptor reduces the exacerbation in said subject. 70-86. (canceled)
 87. A method of inhibiting an inflammatory response or condition in a subject comprising administering to said subject an effective amount of an agent that inhibits CXCL13 activity, wherein the inhibition of CXCL13 activity inhibits the inflammatory response in said subject. 88-104. (canceled)
 105. The method of claim 87, wherein said inflammatory response is a T cell-mediated inflammatory response or condition. 106-124. (canceled)
 125. The method of claim 87, wherein said agent inhibits CXCL13 activity by blocking interaction of CXCL13 with a receptor. 126-130. (canceled)
 131. The method of claim 87, wherein said agent comprises an antibody that specifically binds CXCR5.
 132. (canceled)
 133. The method of claim 87, wherein said agent is an antisense oligonucleotide or an siRNA molecule that inhibits expression of CXCL13.
 134. The method of claim 87, wherein said inhibition of CXCL13 inhibits extralymphatic lymphoid neogenesis in said subject.
 135. The method of claim 87, wherein said inhibition of CXCL13 inhibits recruitment of T_(em) cells or activated B cells into a CNS inflammatory site in said subject.
 136. The method of claim 87, wherein said inhibition of CXCL13 activity inhibits B cell activation or differentiation in a CNS inflammatory site in said subject.
 137. The method of claim 87, wherein said inhibition of CXCL13 action inhibits the migration of CD4+CXCR5+ T_(em) cells or CXCR5+ B cells from the periphery into the CNS in said subject. 